Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

NASA Astrophysics Data System (ADS)

Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

2014-09-01

Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

Use of a benzimidazole derivative BF-188 in fluorescence multispectral imaging for selective visualization of tau protein fibrils in the Alzheimer's disease brain.

PubMed

Harada, Ryuichi; Okamura, Nobuyuki; Furumoto, Shozo; Yoshikawa, Takeo; Arai, Hiroyuki; Yanai, Kazuhiko; Kudo, Yukitsuka

2014-02-01

Selective visualization of amyloid-β and tau protein deposits will help to understand the pathophysiology of Alzheimer's disease (AD). Here, we introduce a novel fluorescent probe that can distinguish between these two deposits by multispectral fluorescence imaging technique. Fluorescence spectral analysis was performed using AD brain sections stained with novel fluorescence compounds. Competitive binding assay using [(3)H]-PiB was performed to evaluate the binding affinity of BF-188 for synthetic amyloid-β (Aβ) and tau fibrils. In AD brain sections, BF-188 clearly stained Aβ and tau protein deposits with different fluorescence spectra. In vitro binding assays indicated that BF-188 bound to both amyloid-β and tau fibrils with high affinity (K i  < 10 nM). In addition, BF-188 showed an excellent blood-brain barrier permeability in mice. Multispectral imaging with BF-188 could potentially be used for selective in vivo imaging of tau deposits as well as amyloid-β in the brain.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE PAGES

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

2017-11-24

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

PubMed

Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

2005-10-01

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

Fluorescence Quenching Property of C-Phycocyanin from Spirulina platensis and its Binding Efficacy with Viable Cell Components.

PubMed

Paswan, Meenakshi B; Chudasama, Meghna M; Mitra, Madhusree; Bhayani, Khushbu; George, Basil; Chatterjee, Shruti; Mishra, Sandhya

2016-03-01

Phycocyanin is a natural brilliant blue colored, fluorescent protein, which is commonly present in cyanobacteria. In this study, C-phycocyanin was extracted and purified from Spirulina platensis, which are multicellular and filamentous cyanobacteria of greater importance because of its various biological and pharmacological potential. It was analyzed for its binding affinity towards blood cells, algal cells, genomic DNA of microalgae, and bacteria at different temperature and incubation time. It showed good binding affinity with these components even at low concentration of 2.5 μM. The purpose of this study was to evaluate the applicability of C-phycocyanin as a green fluorescent dye substituting carcinogenic chemical dyes.

Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

ERIC Educational Resources Information Center

Wang, Sheila C.; Zamble, Deborah B.

2006-01-01

A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ...

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

A Fluorescence Polarization Biophysical Assay for the Naegleria DNA Hydroxylase Tet1.

PubMed

Marholz, Laura J; Wang, Wei; Zheng, Yu; Wang, Xiang

2016-02-11

The discovery of the 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (Tet) protein family was an important advancement in our understanding of DNA-modified epigenetics. Potent inhibitors of these proteins are greatly desired for both the understanding of the functions of these enzymes and to serve as eventual therapeutic leads. So far, the discovery of such small molecules with high affinity has been quite limited. Original tools to screen for activity are greatly needed in order to accelerate this process. Here we present a novel fluorescent probe, and the results of a fluorescence polarization-based binding assay for Naegleria Tet1, a homologue to mammalian Tet. A fluorescence polarization-based competition assay was also established and applied to the rapid and quantitative measurement of the binding affinity of the cofactor αKG and several known Tet1 inhibitors.

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

NASA Astrophysics Data System (ADS)

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants ( Ka) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02 × 10 7 and 2.07 × 10 4 L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S 0 → S 1 transition of esculin ( λexmax≈340 nm) appears, which is similar to the λemmax of BSA and HSA. The critical distance ( R0) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method.

PubMed

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K(a)) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02x10(7) and 2.07x10(4)L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S(0)-->S(1) transition of esculin (lambda(ex)(max) approximately 340nm) appears, which is similar to the lambda(em)(max) of BSA and HSA. The critical distance (R(0)) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

Analysis of Carbohydrate-Carbohydrate Interactions Using Sugar-Functionalized Silicon Nanoparticles for Cell Imaging.

PubMed

Lai, Chian-Hui; Hütter, Julia; Hsu, Chien-Wei; Tanaka, Hidenori; Varela-Aramburu, Silvia; De Cola, Luisa; Lepenies, Bernd; Seeberger, Peter H

2016-01-13

Protein-carbohydrate binding depends on multivalent ligand display that is even more important for low affinity carbohydrate-carbohydrate interactions. Detection and analysis of these low affinity multivalent binding events are technically challenging. We describe the synthesis of dual-fluorescent sugar-capped silicon nanoparticles that proved to be an attractive tool for the analysis of low affinity interactions. These ultrasmall NPs with sizes of around 4 nm can be used for NMR quantification of coupled sugars. The silicon nanoparticles are employed to measure the interaction between the cancer-associated glycosphingolipids GM3 and Gg3 and the associated kD value by surface plasmon resonance experiments. Cell binding studies, to investigate the biological relevance of these carbohydrate-carbohydrate interactions, also benefit from these fluorescent sugar-capped nanoparticles.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

PubMed

Trachman, Robert J; Abdolahzadeh, Amir; Andreoni, Alessio; Cojocaru, Razvan; Knutson, Jay R; Ryckelynck, Michael; Unrau, Peter J; Ferré-D'Amaré, Adrian R

2018-05-24

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

A red-emitting indolium fluorescence probe for membranes - flavonoids interactions.

PubMed

Gao, Qingyun; Liu, Han; Ding, Qiongjie; Du, Jinya; Liu, Chunlin; Yang, Wei; Shen, Ping; Yang, Changying

2018-05-01

The red-emitting indolium derivative compound (E)-2-(4-(diphenylamino)styryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (H3) was demonstrated as a sensitive membrane fluorescence probe. The probe located at the interface of liposomes when mixed showed much fluorescence enhancement by inhibiting the twisted intramolecular charge transfer state. After ultrasonic treatment, it penetrated into lipid bilayers with the emissions leveling off and a rather large encapsulation efficiency (71.4%) in liposomes. The ζ-potential and particle size measurement confirmed that the charged indolium group was embedded deeply into lipid bilayers. The probe was then used to monitor the affinities of antioxidant flavonoids for membranes. It was verified that quercetin easily interacted with liposomes and dissociated the probe from the internal lipid within 60 s under the condition of simply mixing. The assessment of binding affinities of six flavonoids and the coincident results with their antioxidation activities indicated that it was a promising membrane probe for the study of drug bio-affinities. Copyright © 2018 John Wiley & Sons, Ltd.

Analysis of conjugation of chloramphenicol and hemoglobin by fluorescence, circular dichroism and molecular modeling

NASA Astrophysics Data System (ADS)

Ding, Fei; Liu, Wei; Sun, Ye; Yang, Xin-Ling; Sun, Ying; Zhang, Li

2012-01-01

Chloramphenicol is a low cost, broad spectrum, highly active antibiotic, and widely used in the treatment of serious infections, including typhoid fever and other life-threatening infections of the central nervous system and respiratory tract. The purpose of the present study was to examine the conjugation of chloramphenicol with hemoglobin (Hb) and compared with albumin at molecular level, utilizing fluorescence, UV/vis absorption, circular dichroism (CD) as well as molecular modeling. Fluorescence data indicate that drug bind Hb generate quenching via static mechanism, this corroborates UV/vis absorption measurements that the ground state complex formation with an affinity of 10 4 M -1, and the driving forces in the Hb-drug complex are hydrophilic interactions and hydrogen bonds, as derived from computational model. The accurate binding site of drug has been identified from the analysis of fluorescence and molecular modeling, α1β2 interface of Hb was assigned to possess high-affinity for drug, which located at the β-37 Trp nearby. The structural investigation of the complexed Hb by synchronous fluorescence, UV/vis absorption, and CD observations revealed some degree of Hb structure unfolding upon complexation. Based on molecular modeling, we can draw the conclusion that the binding affinity of drug with albumin is superior, compared with Hb. These phenomena can provide salient information on the absorption, distribution, pharmacology, and toxicity of chloramphenicol and other drugs which have analogous configuration with chloramphenicol.

Fluorescent nanoscale zinc(II)-carboxylate coordination polymers for explosive sensing.

PubMed

Zhang, Chengyi; Che, Yanke; Zhang, Zengxing; Yang, Xiaomei; Zang, Ling

2011-02-28

Fluorescent nanoscale coordination polymers with cubic morphology and long range ordered structure were fabricated and exhibited efficient sensing for both nitroaromatic explosive and nitromethane due to large surface area to volume ratio and strong binding affinity to explosive molecules.

The Leading Group Effect: Illusionary Declines in Scholastic Standard Scores of Mid-Range Japanese Junior High School Pupils

ERIC Educational Resources Information Center

Mori, Kazuo; Uchida, Akitoshi

2012-01-01

Longitudinal change in the average Z scores for four groups of pupils sorted by quartiles was examined for its stability over three years. The data, collected from 1998 to 2009, was obtained from nine cohorts of Japanese junior high school pupils totaling 1,962 subjects. It showed illusionary declines among the mid-range pupils but improvements…

A Longitudinal Study of the Six Degrees of Freedom Cervical Spine Range of Motion During Dynamic Flexion/Extension and Rotation After Single-Level Anterior Arthrodesis

PubMed Central

Anderst, William J.; West, Tyler; Donaldson, William F; Lee, Joon Y.; Kang, James D.

2016-01-01

Study Design A longitudinal study using biplane radiography to measure in vivo intervertebral range of motion (ROM) during dynamic flexion/extension and rotation. Objective To longitudinally compare intervertebral maximal ROM and midrange motion in asymptomatic control subjects and single-level arthrodesis patients. Summary of Background Data In vitro studies consistently report that adjacent segment maximal ROM increases superior and inferior to cervical arthrodesis. Previous in vivo results have been conflicting, indicating that maximal ROM may or may not increase superior and/or inferior to the arthrodesis. There are no previous reports of midrange motion in arthrodesis patients and similar-aged controls. Methods Eight single-level (C5/C6) anterior arthrodesis patients (tested 7±1 months and 28±6 months post-surgery) and six asymptomatic control subjects (tested twice, 58±6 months apart) performed dynamic full ROM flexion/extension and axial rotation while biplane radiographs were collected at 30 images/s. A previously validated tracking process determined three-dimensional vertebral position from each pair of radiographs with sub-millimeter accuracy. The intervertebral maximal ROM and midrange motion in flexion/extension, rotation, lateral bending, and anterior-posterior translation were compared between test dates and between groups. Results Adjacent segment maximal ROM did not increase over time during flexion/extension or rotation movements. Adjacent segment maximal rotational ROM was not significantly greater in arthrodesis patients than in corresponding motion segments of similar-aged controls. C4/C5 adjacent segment rotation during the midrange of head motion and maximal anterior-posterior translation were significantly greater in arthrodesis patients than in the corresponding motion segment in controls on the second test date. Conclusions C5/C6 arthrodesis appears to significantly affect midrange, but not end-range, adjacent segment motions. The effects of arthrodesis on adjacent segment motion may be best evaluated by longitudinal studies that compare maximal and midrange adjacent segment motion to corresponding motion segments of similar-aged controls to determine if the adjacent segment motion is truly excessive. PMID:27831986

Longitudinal Study of the Six Degrees of Freedom Cervical Spine Range of Motion During Dynamic Flexion, Extension, and Rotation After Single-level Anterior Arthrodesis.

PubMed

Anderst, William J; West, Tyler; Donaldson, William F; Lee, Joon Y; Kang, James D

2016-11-15

A longitudinal study using biplane radiography to measure in vivo intervertebral range of motion (ROM) during dynamic flexion/extension, and rotation. To longitudinally compare intervertebral maximal ROM and midrange motion in asymptomatic control subjects and single-level arthrodesis patients. In vitro studies consistently report that adjacent segment maximal ROM increases superior and inferior to cervical arthrodesis. Previous in vivo results have been conflicting, indicating that maximal ROM may or may not increase superior and/or inferior to the arthrodesis. There are no previous reports of midrange motion in arthrodesis patients and similar-aged controls. Eight single-level (C5/C6) anterior arthrodesis patients (tested 7 ± 1 months and 28 ± 6 months postsurgery) and six asymptomatic control subjects (tested twice, 58 ± 6 months apart) performed dynamic full ROM flexion/extension and axial rotation whereas biplane radiographs were collected at 30 images per second. A previously validated tracking process determined three-dimensional vertebral position from each pair of radiographs with submillimeter accuracy. The intervertebral maximal ROM and midrange motion in flexion/extension, rotation, lateral bending, and anterior-posterior translation were compared between test dates and between groups. Adjacent segment maximal ROM did not increase over time during flexion/extension, or rotation movements. Adjacent segment maximal rotational ROM was not significantly greater in arthrodesis patients than in corresponding motion segments of similar-aged controls. C4/C5 adjacent segment rotation during the midrange of head motion and maximal anterior-posterior translation were significantly greater in arthrodesis patients than in the corresponding motion segment in controls on the second test date. C5/C6 arthrodesis appears to significantly affect midrange, but not end-range, adjacent segment motions. The effects of arthrodesis on adjacent segment motion may be best evaluated by longitudinal studies that compare maximal and midrange adjacent segment motion to corresponding motion segments of similar-aged controls to determine if the adjacent segment motion is truly excessive. 3.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

PubMed

Buku, Angeliki; Mendlowitz, Milton; Condie, Barry A; Price, Joseph A

2004-06-01

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

PubMed Central

2013-01-01

Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient. PMID:23687945

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

PubMed

Zimmermann, Gunther; Li, Yizhou; Rieder, Ulrike; Mattarella, Martin; Neri, Dario; Scheuermann, Jörg

2017-05-04

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diurnal Solar Energy Conversion and Photoprotection in Rice Canopies1[OPEN

PubMed Central

Quick, W. Paul; von Caemmerer, Susanne; Furbank, Robert

2017-01-01

Genetic improvement of photosynthetic performance of cereal crops and increasing the efficiency with which solar radiation is converted into biomass has recently become a major focus for crop physiologists and breeders. The pulse amplitude modulated chlorophyll fluorescence technique (PAM) allows quantitative leaf level monitoring of the utilization of energy for photochemical light conversion and photoprotection in natural environments, potentially over the entire crop lifecycle. Here, the diurnal relationship between electron transport rate (ETR) and irradiance was measured in five cultivars of rice (Oryza sativa) in canopy conditions with PAM fluorescence under natural solar radiation. This relationship differed substantially from that observed for conventional short term light response curves measured under controlled actinic light with the same leaves. This difference was characterized by a reduced curvature factor when curve fitting was used to model this diurnal response. The engagement of photoprotective processes in chloroplast electron transport in leaves under canopy solar radiation was shown to be a major contributor to this difference. Genotypic variation in the irradiance at which energy flux into photoprotective dissipation became greater than ETR was observed. Cultivars capable of higher ETR at midrange light intensities were shown to produce greater leaf area over time, estimated by noninvasive imaging. PMID:27895208

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

PubMed

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

Potential toxicity and affinity of triphenylmethane dye malachite green to lysozyme.

PubMed

Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Ma, Lin; Yang, Xin-Ling; Zhang, Li; Sun, Ying

2012-04-01

Malachite green is a triphenylmethane dye that is used extensively in many industrial and aquacultural processes, generating environmental concerns and health problems to human being. In this contribution, the complexation between lysozyme and malachite green was verified by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) approaches. The precise binding patch of malachite green in lysozyme has been identified from molecular modeling and ANS displacement, Trp-62, Trp-63, and Trp-108 residues of lysozyme were earmarked to possess high-affinity for this dye, the principal forces in the lysozyme-malachite green adduct are hydrophobic and π-π interactions. Steady state fluorescence proclaimed the complex of malachite green with lysozyme yields quenching through static type, which substantiates time-resolved fluorescence measurements that lysozyme-malachite green conjugation formation has an affinity of 10(3)M(-1). Moreover, via molecular modeling and also CD data, we can safely arrive at a conclusion that the polypeptide chain of lysozyme partially destabilized upon complexation with malachite green. The data emerged here will help to further understand the toxicological action of malachite green in human body. Copyright © 2012 Elsevier Inc. All rights reserved.

Using mid-range laser scanners to digitize cultural-heritage sites.

PubMed

Spring, Adam P; Peters, Caradoc; Minns, Tom

2010-01-01

Here, we explore new, more accessible ways of modeling 3D data sets that both professionals and amateurs can employ in areas such as architecture, forensics, geotechnics, cultural heritage, and even hobbyist modeling. To support our arguments, we present images from a recent case study in digital preservation of cultural heritage using a mid-range laser scanner. Our appreciation of the increasing variety of methods for capturing 3D spatial data inspired our research. Available methods include photogrammetry, airborne lidar, sonar, total stations (a combined electronic and optical survey instrument), and midand close-range scanning.1 They all can produce point clouds of varying density. In our case study, the point cloud produced by a mid-range scanner demonstrates how open source software can make modeling and disseminating data easier. Normally, researchers would model this data using expensive specialized software, and the data wouldn't extend beyond the laser-scanning community.

Fluorescence/depolarization lidar for mid-range stand-off detection of biological agents

NASA Astrophysics Data System (ADS)

Mierczyk, Z.; Kopczyński, K.; Zygmunt, M.; Wojtanowski, J.; Młynczak, J.; Gawlikowski, A.; Młodzianko, A.; Piotrowski, W.; Gietka, A.; Knysak, P.; Drozd, T.; Muzal, M.; Kaszczuk, M.; Ostrowski, R.; Jakubaszek, M.

2011-06-01

LIDAR system for real-time standoff detection of bio-agents is presented and preliminary experimental results are discussed. The detection approach is based on two independent physical phenomena: (1) laser induced fluorescence (LIF), (2) depolarization resulting from elastic scattering on non-spherical particles. The device includes three laser sources, two receiving telescopes, depolarization component and spectral signature analyzing spectrograph. It was designed to provide the stand-off detection capability at ranges from 200 m up to several kilometers. The system as a whole forms a mobile platform for vehicle or building installation. Additionally, it's combined with a scanning mechanics and advanced software, which enable to conduct the semi-automatic monitoring of a specified space sector. For fluorescence excitation, 3-rd (355 nm) and 4-th (266 nm) harmonics of Nd:YAG pulsed lasers are used. They emit short (~6 ns) pulses with the repetition rate of 20 Hz. Collecting optics for fluorescence echo detection and spectral content analysis includes 25 mm diameter f/4 Newton telescope, Czerny Turner spectrograph and 32-channel PMT. Depending on the grating applied, the spectral resolution from 20 nm up to 3 nm per channel can be achieved. The system is also equipped with an eye-safe (1.5 μm) Nd:YAG OPO laser for elastic backscattering/depolarization detection. The optical echo signal is collected by Cassegrain telescope with aperture diameter of 12.5 mm. Depolarization detection component based on polarizing beam-splitter serves as the stand-off particle-shape analyzer, which is very valuable in case of non-spherical bio-aerosols sensing.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

PubMed

Gallo, Eugenio; Jarvik, Jonathan W

2017-08-01

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications. © 2017. Published by The Company of Biologists Ltd.

The Unevenness and Non-orthogonal State of Distribution of Corneal Thickness and the Influence on Correction of Myopic Astigmatism by LASEK.

PubMed

Wang, Shulin; Wang, Xin; Liu, Mingna; Wang, Haiying; Li, Jing; Shi, Weiyun

2015-09-01

To observe and calculate the unevenness and the non-orthogonal state of distribution of corneal thickness and the relationship between them using Pentacam and to investigate the influence of unevenness and the non-orthogonal state on correction of myopic astigmatism by laser subepithelial keratomileusis (LASEK). 230 eyes with myopic astigmatism treated with LASEK were divided into two groups: 114 eyes as the low astigmatism group (-0.25 to -0.75 DC) and 116 eyes as the midrange-high astigmatism group (-1.00 to -4.50 DC). With the help of the diagram of keratoconus evaluation program of the Pentacam, the D 3.0 and D 6.5 were calculated for the index of distribution of unevenness of the corneal thickness, and the absolute value of the angle between the maximum and minimum progression-index orientation (M 90) for the index of non-orthogonal states. The correction of myopic astigmatism by LASEK was based on standard vector analysis and power vector analysis. The follow-up period was for 3 months. The preoperative M 90 was 22.14° ± 20.87°, D 6.5 was 58.66 ± 21.32 μm, and D 3.0 was 16.11 ± 4.28 μm for the 230 eyes that were tested. The D 6.5 of low astigmatism group (55.62 ± 20.81) μm was significantly lower than that of midrange-high astigmatism group (61.65 ± 21.48) μm (P < 0.05). Of the 230 eyes, the M 90 was positively correlated with D 6.5 (r = 0.37, P < 0.001), and D 6.5 was positively correlated with D 3.0 (r = 0.56, P < 0.001). 3 months postoperatively, the absolute error vector (|EV|) of low astigmatism group (0.46 ± 0.34) was significantly lower than that of midrange-high astigmatism group (0.53 ± 0.29) (P < 0.01). The error of magnitude of low astigmatism group (-0.10 ± 0.31) was significantly lower than that of midrange-high astigmatism group (0.08 ± 0.41) (P < 0.001). The absolute error of angle (|EA|) of low astigmatism group (26.10 ± 27.24) was significantly higher than that of midrange-high astigmatism group (9.99 ± 17.32) (P < 0.001). The correction ratio of low astigmatism group (1.45 ± 1.21) was significantly higher than that of midrange-high astigmatism group (0.94 ± 0.33) (P < 0.01). The error ratio (ER) of low astigmatism group (1.34 ± 1.40) was significantly higher than that of midrange-high astigmatism group (0.42 ± 0.27) (P < 0.001). In low astigmatism group, M 90 was positively correlated with |EV| (r = 0.30, P < 0.001). In midrange-high astigmatism group, M 90 was positively correlated with ER (r = 0.31, P < 0.001) and D 6.5 was positively correlated with |EV| and B, respectively (r = 0.34, 0.33, P < 0.001). The relationship between unevenness and non-orthogonal state of distribution of corneal thickness could influence the correction of astigmatism by LASEK. Therefore, more attention should be paid to the correction of midrange-high astigmatism group by excimer surgery.

Current use was established and Cochrane guidance on selection of social theories for systematic reviews of complex interventions was developed.

PubMed

Noyes, Jane; Hendry, Maggie; Booth, Andrew; Chandler, Jackie; Lewin, Simon; Glenton, Claire; Garside, Ruth

2016-07-01

To identify examples of how social theories are used in systematic reviews of complex interventions to inform production of Cochrane guidance. Secondary analysis of published/unpublished examples of theories of social phenomena for use in reviews of complex interventions identified through scoping searches, engagement with key authors and methodologists supplemented by snowballing and reference searching. Theories were classified (low-level, mid-range, grand). Over 100 theories were identified with evidence of proliferation over the last 5 years. New low-level theories (tools, taxonomies, etc) have been developed for classifying and reporting complex interventions. Numerous mid-range theories are used; one example demonstrated how control theory had changed the review's findings. Review-specific logic models are increasingly used, but these can be challenging to develop. New low-level and mid-range psychological theories of behavior change are evolving. No reviews using grand theory (e.g., feminist theory) were identified. We produced a searchable Wiki, Mendeley Inventory, and Cochrane guidance. Use of low-level theory is common and evolving; incorporation of mid-range theory is still the exception rather than the norm. Methodological work is needed to evaluate the contribution of theory. Choice of theory reflects personal preference; application of theory is a skilled endeavor. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

PubMed Central

Ho, Cheng-Hsun; Frommer, Wolf B

2014-01-01

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. DOI: http://dx.doi.org/10.7554/eLife.01917.001 PMID:24623305

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin.

PubMed

Xu, Yujing; Hong, Tingting; Chen, Xueping; Ji, Yibing

2017-05-01

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S-omeprazole, S-OME) and its R-enantiomer (R-omeprazole, R-OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10 3 M -1 and 5.36 × 10 3 M -1 , respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

PubMed

DeJong, Eric S; Chang, Chia-en; Gilson, Michael K; Marino, John P

2003-07-08

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.

CdS/TiO2-fluorescein isothiocyanate nanoparticles as fluorescence resonance energy transfer probe for the determination of trace alkaline phosphatase based on affinity adsorption assay.

PubMed

Liu, Jia-Ming; Lin, Li-ping; Jiao, Li; Cui, Ma-Lin; Wang, Xin-Xing; Zhang, Li-Hong; Zheng, Zhi-Yong

2012-08-30

The CdS/TiO(2)-fluorescein isothiocyanate (FITC) luminescent nanoparticles (CdS/TiO(2)-FITC) with the particle size of 20 nm have been synthesized by sol-gel method. CdS/TiO(2)-FITC could emit the fluorescence of both FITC and CdS/TiO(2). The fluorescence resonance energy transfer (FRET) occurred between the donor CdS/TiO(2) and the acceptor FITC in the CdS/TiO(2)-FITC. Taking advantages of the excellent characteristics of FRET, a new CdS/TiO(2)-FITC FRET labeling reagent and a CdS/TiO(2)-FITC-wheat germ agglutinin (CdS/TiO(2)-FITC-WGA) fluorescent probe have been developed. The FRET occurring between the donor CdS/TiO(2) and the acceptor FITC in the labelled product CdS/TiO(2)-FITC-WGA-AP, formed in the affinity adsorption reaction between the WGA in this CdS/TiO(2)-FITC-WGA fluorescent probe and alkaline phosphatase (AP), sharply enhanced the fluorescence signal of FITC and quench the fluorescence signal of CdS/TiO(2). Moreover, the ΔF (the change of the fluorescence signal) of FITC and CdS/TiO(2) were proportional to the content of AP, respectively. Thus, a new method that CdS/TiO(2)-fluorescein isothiocyanate nanoparticles for the determination of trace AP based on FRET-affinity adsorption assay has been established. The limit of quantification (LOQ) of the method was 1.3×10(-17) g AP mL(-1) for CdS/TiO(2) and 1.1×10(-17) g AP mL(-1) for FITC, respectively. This sensitive, rapid, high selective and precise method has been applied to the determination of AP in human serum and the prediction of human disease with the results agreed well with enzyme-linked immunosorbent assay (ELISA) in Zhangzhou Municipal Hospital of Fujian Province. Simultaneously, the reaction mechanism for the determination of AP was also discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

A Complex Story: Universal Preference vs. Individual Differences Shaping Aesthetic Response to Fractals Patterns.

PubMed

Street, Nichola; Forsythe, Alexandra M; Reilly, Ronan; Taylor, Richard; Helmy, Mai S

2016-01-01

Fractal patterns offer one way to represent the rough complexity of the natural world. Whilst they dominate many of our visual experiences in nature, little large-scale perceptual research has been done to explore how we respond aesthetically to these patterns. Previous research (Taylor et al., 2011) suggests that the fractal patterns with mid-range fractal dimensions (FDs) have universal aesthetic appeal. Perceptual and aesthetic responses to visual complexity have been more varied with findings suggesting both linear (Forsythe et al., 2011) and curvilinear (Berlyne, 1970) relationships. Individual differences have been found to account for many of the differences we see in aesthetic responses but some, such as culture, have received little attention within the fractal and complexity research fields. This two-study article aims to test preference responses to FD and visual complexity, using a large cohort (N = 443) of participants from around the world to allow universality claims to be tested. It explores the extent to which age, culture and gender can predict our preferences for fractally complex patterns. Following exploratory analysis that found strong correlations between FD and visual complexity, a series of linear mixed-effect models were implemented to explore if each of the individual variables could predict preference. The first tested a linear complexity model (likelihood of selecting the more complex image from the pair of images) and the second a mid-range FD model (likelihood of selecting an image within mid-range). Results show that individual differences can reliably predict preferences for complexity across culture, gender and age. However, in fitting with current findings the mid-range models show greater consistency in preference not mediated by gender, age or culture. This article supports the established theory that the mid-range fractal patterns appear to be a universal construct underlying preference but also highlights the fragility of universal claims by demonstrating individual differences in preference for the interrelated concept of visual complexity. This highlights a current stalemate in the field of empirical aesthetics.

Introducing a fluorescence-based standard to quantify protein partitioning into membranes.

PubMed

Thomas, Franziska A; Visco, Ilaria; Petrášek, Zdeněk; Heinemann, Fabian; Schwille, Petra

2015-11-01

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface. Copyright © 2015. Published by Elsevier B.V.

Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1998-01-01

Novel fluorescent heterodimeric DNA-staining energy transfer dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts. Kits and individual compounds are provided, where the kits find use for simultaneous detection of a variety of moieties, particularly using a single narrow wavelength irradiation source. The individual compounds are characterized by high donor quenching and high affinity to dsDNA as a result of optimizing the length of the linking group separating the two chromophores.

Novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies.

PubMed

Wright, Michael; Miller, Andrew D

2006-02-15

Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.

Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.

PubMed

Zhang, Zijie; Liu, Juewen

2016-03-01

Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

NASA Technical Reports Server (NTRS)

Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

2002-01-01

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

PubMed

Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M

2018-06-01

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.

Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

NASA Astrophysics Data System (ADS)

Shu, Chang; Ding, Li; Zhong, Wenying

2014-10-01

In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

Method and apparatus for detection of fluorescently labeled materials

DOEpatents

Stern, David; Fiekowsky, Peter

2004-05-25

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

NASA Astrophysics Data System (ADS)

Zhang, Congying; Gu, Yueqing

2014-09-01

Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

PubMed

Collot, Mayeul; Loukou, Christina; Yakovlev, Aleksey V; Wilms, Christian D; Li, Dongdong; Evrard, Alexis; Zamaleeva, Alsu; Bourdieu, Laurent; Léger, Jean-François; Ropert, Nicole; Eilers, Jens; Oheim, Martin; Feltz, Anne; Mallet, Jean-Maurice

2012-09-12

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

ERIC Educational Resources Information Center

Hicks, Katherine A.

2016-01-01

Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1996-01-01

Novel fluorescent DNA-staining dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts.

Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

PubMed Central

2013-01-01

Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741. PMID:23668665

Binding patterns and structure-affinity relationships of food azo dyes with lysozyme: a multitechnique approach.

PubMed

Peng, Wei; Ding, Fei; Peng, Yu-Kui; Jiang, Yu-Ting; Zhang, Li

2013-12-18

Food dyes serve to beguile consumers: they are often used to imitate the presence of healthful, colorful food produce such as fruits and vegetables. But considering the hurtful impact of these chemicals on the human body, it is time to thoroughly uncover the toxicity of these food dyes at the molecular level. In the present contribution, we have examined the molecular reactions of protein lysozyme with model food azo compound Color Index (C.I.) Acid Red 2 and its analogues C.I. Acid Orange 52, Solvent Yellow 2, and the core structure of azobenzene using a combination of biophysical methods at physiological conditions. Fluorescence, circular dichroism (CD), time-resolved fluorescence, UV-vis absorption as well as computer-aided molecular modeling were used to analyze food dye affinity, binding mode, energy transfer, and the effects of food dye complexation on lysozyme stability and conformation. Fluorescence emission spectra indicate complex formation at 10(-5) M dye concentration, and this corroborates time-resolved fluorescence results showing the diminution in the tryptophan (Trp) fluorescence mainly via a static type (KSV = 1.505 × 10(4) M(-1)) and Förster energy transfer. Structural analysis displayed the participation of several amino acid residues in food dye protein adducts, with hydrogen bonds, π-π and cation-π interactions, but the conformation of lysozyme was unchanged in the process, as derived from fluorescence emission, far-UV CD, and synchronous fluorescence spectra. The overall affinity of food dye is 10(4) M(-1) and there exists only one kind of binding domain in protein for food dye. These data are consistent with hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, and molecular modeling manifesting the food dye binding patch was near to Trp-62 and Trp-63 residues of lysozyme. On the basis of the computational analyses, we determine that the type of substituent on the azobenzene structure has a powerful influence on the toxicity of food dyes. Results from this work testify that model protein, though an indirect method, provides a more comprehensive profile of the essence of toxicity evaluation of food dyes.

Human serum albumin stability and toxicity of anthraquinone dye alizarin complexone: an albumin-dye model.

PubMed

Ding, Fei; Zhang, Li; Diao, Jian-Xiong; Li, Xiu-Nan; Ma, Lin; Sun, Ying

2012-05-01

The complexation between the primary vector of ligands in blood plasma, human serum albumin (HSA) and a toxic anthraquinone dye alizarin complexone, was unmasked by means of circular dichroism (CD), molecular modeling, steady state and time-resolved fluorescence, and UV/vis absorption measurements. The structural investigation of the complexed HSA through far-UV CD, three-dimensional and synchronous fluorescence shown the polypeptide chain of HSA partially destabilizing with a reduction of α-helix upon conjugation. From molecular modeling and competitive ligand binding results, Sudlow's site I, which was the same as that of warfarin-azapropazone site, was appointed to retain high-affinity for alizarin complexone. Moreover, steady state fluorescence displayed that static type and Förster energy transfer is the operational mechanism for the vanish in the tryptophan (Trp)-214 fluorescence, this corroborates time-resolved fluorescence that HSA-alizarin complexone adduct formation has an affinity of 10(5) M(-1), and the driving forces were found to be chiefly π-π, hydrophobic, and hydrogen bonds, associated with an exothermic free energy change. These data should be utilized to illustrate the mechanism by which the toxicological action of anthraquinone dyes is mitigated by transporter HSA. Copyright © 2012 Elsevier Inc. All rights reserved.

Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

PubMed

Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

2015-02-09

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

PubMed Central

Faurobert, E; Otto-Bruc, A; Chardin, P; Chabre, M

1993-01-01

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding. Images PMID:8223434

NATO’S Southern Flank: A Strategic Assessment.

DTIC Science & Technology

The study conducts a critical evaluation of the Southern Flank members of NATO (Italy, Greece, and Turkey), to include the political, cultural, and economic stability of each, as well as the capability of their armed forces to contribute to the NATO mission, and within this context, to assess the NATO mid-range strategy for the ’Southern Region.’ A brief assessment was also made of the entire Mediterranean littoral as well as other factors that might influence the mid-range strategy, e.g., the Arab-Israeli conflict, MBFR, etc.

Laser range measurement for a satellite navigation scheme and mid-range path selection and obstacle avoidance. M.S. Thesis

NASA Technical Reports Server (NTRS)

Zuraski, G. D.

1972-01-01

The functions of a laser rangefinder on board an autonomous Martian roving vehicle are discussed. The functions are: (1) navigation by means of a passive satellite and (2) mid-range path selection and obstacle avoidance. The feasibility of using a laser to make the necessary range measurements is explored and a preliminary design is presented. The two uses of the rangefinder dictate widely different operating parameters making it impossible to use the same system for both functions.

Diurnal Solar Energy Conversion and Photoprotection in Rice Canopies.

PubMed

Meacham, Katherine; Sirault, Xavier; Quick, W Paul; von Caemmerer, Susanne; Furbank, Robert

2017-01-01

Genetic improvement of photosynthetic performance of cereal crops and increasing the efficiency with which solar radiation is converted into biomass has recently become a major focus for crop physiologists and breeders. The pulse amplitude modulated chlorophyll fluorescence technique (PAM) allows quantitative leaf level monitoring of the utilization of energy for photochemical light conversion and photoprotection in natural environments, potentially over the entire crop lifecycle. Here, the diurnal relationship between electron transport rate (ETR) and irradiance was measured in five cultivars of rice (Oryza sativa) in canopy conditions with PAM fluorescence under natural solar radiation. This relationship differed substantially from that observed for conventional short term light response curves measured under controlled actinic light with the same leaves. This difference was characterized by a reduced curvature factor when curve fitting was used to model this diurnal response. The engagement of photoprotective processes in chloroplast electron transport in leaves under canopy solar radiation was shown to be a major contributor to this difference. Genotypic variation in the irradiance at which energy flux into photoprotective dissipation became greater than ETR was observed. Cultivars capable of higher ETR at midrange light intensities were shown to produce greater leaf area over time, estimated by noninvasive imaging. © 2017 American Society of Plant Biologists. All Rights Reserved.

Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

PubMed

Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

2018-05-15

Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

PubMed

Luzy, Jean-Philippe; Chen, Huixiong; Gril, Brunilde; Liu, Wang-Qing; Vidal, Michel; Perdereau, Dominique; Burnol, Anne-Françoise; Garbay, Christiane

2008-02-01

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

Rapid, Sensitive Detection of Botulinum Toxin on a Flexible Microfluidics Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Dockendorff, Brian P.; Feldhaus, Michael J.

2004-10-30

In this paper we will describe how high affinity reagents and a sensor configuration enabling rapid mass transport can be combined for rapid, sensitive biodetection. The system that we have developed includes a renewable surface immunoassay, which involves on-column detection of a fluorescently labeled secondary antibody in a sandwich immunoassay. Yeast display and directed molecular evolution were used to create high affinity antibodies to the botulinum toxin heavy chain receptor binding domain, AR1 and 3D12. A rotating rod renewable surface microcolumn was used to form a microliter-sized column containing beads functionalized with the capture antibody (AR1). The column was perfusedmore » with sample, wash solutions, and a fluorescently labeled secondary antibody (3D12) while the on-column fluorescence was monitored. Detection was accomplished in less than 5 minutes, with a total processing time of about 10 minutes. On-column detection of botulinum toxin was more sensitive and much faster than flow cytometry analysis on microbeads using the same reagents.« less

A highly selective colorimetric and ratiometric fluorescent chemodosimeter for detection of fluoride ions based on 1,8-naphthalimide derivatives.

PubMed

Kai, Yumei; Hu, Yonghong; Wang, Kai; Zhi, Wenbiao; Liang, Mengmeng; Yang, Wenge

2014-01-24

A high selective colorimetric and ratiometric fluorescent probe based on 4-hydroxy-1, 8-naphthalimide was designed and synthesized to detect fluoride ions (F(-)). The sensing behavior of this probe was studied by UV-visible and fluorescence spectroscopy. The probe displays an 110 nm red-shift of fluorescence emission and the color changes from colorless to yellow by virtue of the strong affinity of F(-) toward silicon which can act as a new visual sensor for F(-). Copyright © 2013 Elsevier B.V. All rights reserved.

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

PubMed

Arruda, Maria Augusta; Stoddart, Leigh A; Gherbi, Karolina; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J

2017-01-01

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A 3 AR. These were K114 (pK i 6.43), retinoic acid p -hydroxyanilide (pK i 6.13) and SU 6556 (pK i 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A 3 AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Investigation of the binding affinity in vitamin B12-Bovine serum albumin system using various spectroscopic methods

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2017-09-01

The binding affinity between vitamin B12 (VitB12) and bovine serum albumin (BSA) has been investigated in aqueous solution at pH = 7.4, employing UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative effects noted for BSA intrinsic fluorescence resulting from the interactions with VitB12 confirm the formation of π-π stacked non-covalent and non-fluorescent complexes in the system VitB12-BSA. All the determined parameters, the binding, fluorescence quenching and bimolecular quenching rate constants (of the order of 104 L mol- 1, 103 L mol- 1 and 1011 L mol- 1 s- 1, respectively), as well as Förster resonance energy transfer parameters validate the mechanism of static quenching. The interaction with VitB12 induces folding of the polypeptide chains around Trp residues of BSA, resulting in a more hydrophobic surrounding. Presented outcomes suggest that the addition of VitB12 can lead to the more organized BSA conformation and its more folded tertiary structure, what could influence the physiological functions of bovine serum albumin, notably in case of its overuse or abnormal metabolism.

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

2016-01-01

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

A Complex Story: Universal Preference vs. Individual Differences Shaping Aesthetic Response to Fractals Patterns

PubMed Central

Street, Nichola; Forsythe, Alexandra M.; Reilly, Ronan; Taylor, Richard; Helmy, Mai S.

2016-01-01

Fractal patterns offer one way to represent the rough complexity of the natural world. Whilst they dominate many of our visual experiences in nature, little large-scale perceptual research has been done to explore how we respond aesthetically to these patterns. Previous research (Taylor et al., 2011) suggests that the fractal patterns with mid-range fractal dimensions (FDs) have universal aesthetic appeal. Perceptual and aesthetic responses to visual complexity have been more varied with findings suggesting both linear (Forsythe et al., 2011) and curvilinear (Berlyne, 1970) relationships. Individual differences have been found to account for many of the differences we see in aesthetic responses but some, such as culture, have received little attention within the fractal and complexity research fields. This two-study article aims to test preference responses to FD and visual complexity, using a large cohort (N = 443) of participants from around the world to allow universality claims to be tested. It explores the extent to which age, culture and gender can predict our preferences for fractally complex patterns. Following exploratory analysis that found strong correlations between FD and visual complexity, a series of linear mixed-effect models were implemented to explore if each of the individual variables could predict preference. The first tested a linear complexity model (likelihood of selecting the more complex image from the pair of images) and the second a mid-range FD model (likelihood of selecting an image within mid-range). Results show that individual differences can reliably predict preferences for complexity across culture, gender and age. However, in fitting with current findings the mid-range models show greater consistency in preference not mediated by gender, age or culture. This article supports the established theory that the mid-range fractal patterns appear to be a universal construct underlying preference but also highlights the fragility of universal claims by demonstrating individual differences in preference for the interrelated concept of visual complexity. This highlights a current stalemate in the field of empirical aesthetics. PMID:27252634

Understanding the self-assembly of proteins onto gold nanoparticles and quantum dots driven by metal-histidine coordination.

PubMed

Aldeek, Fadi; Safi, Malak; Zhan, Naiqian; Palui, Goutam; Mattoussi, Hedi

2013-11-26

Coupling of polyhistidine-appended biomolecules to inorganic nanocrystals driven by metal-affinity interactions is a greatly promising strategy to form hybrid bioconjugates. It is simple to implement and can take advantage of the fact that polyhistidine-appended proteins and peptides are routinely prepared using well established molecular engineering techniques. A few groups have shown its effectiveness for coupling proteins onto Zn- or Cd-rich semiconductor quantum dots (QDs). Expanding this conjugation scheme to other metal-rich nanoparticles (NPs) such as AuNPs would be of great interest to researchers actively seeking effective means for interfacing nanostructured materials with biology. In this report, we investigated the metal-affinity driven self-assembly between AuNPs and two engineered proteins, a His7-appended maltose binding protein (MBP-His) and a fluorescent His6-terminated mCherry protein. In particular, we investigated the influence of the capping ligand affinity to the nanoparticle surface, its density, and its lateral extension on the AuNP-protein self-assembly. Affinity gel chromatography was used to test the AuNP-MPB-His7 self-assembly, while NP-to-mCherry-His6 binding was evaluated using fluorescence measurements. We also assessed the kinetics of the self-assembly between AuNPs and proteins in solution, using time-dependent changes in the energy transfer quenching of mCherry fluorescent proteins as they immobilize onto the AuNP surface. This allowed determination of the dissociation rate constant, Kd(-1) ∼ 1-5 nM. Furthermore, a close comparison of the protein self-assembly onto AuNPs or QDs provided additional insights into which parameters control the interactions between imidazoles and metal ions in these systems.

Optimization of the Alkyl Linker of TO Base Surrogate in Triplex-Forming PNA for Enhanced Binding to Double-Stranded RNA.

PubMed

Sato, Takaya; Sato, Yusuke; Nishizawa, Seiichi

2017-03-23

A series of triplex-forming peptide nucleic acid (TFP) probes carrying a thiazole orange (TO) base surrogate through an alkyl linker was synthesized, and the interactions between these so-called tFIT probes and purine-rich sequences within double-stranded RNA (dsRNA) were examined. We found that the TO base surrogate linker significantly affected both the binding affinity and the fluorescence response upon triplex formation with the target dsRNA. Among the probes examined, the TO base surrogate connected through the propyl linker in the tFIT probes increased the binding affinity by a factor of ten while maintaining its function as the fluorescent universal base. Isothermal titration calorimetry experiments revealed that the increased binding affinity resulted from the gain in the binding enthalpy, which could be explained by the enhanced π-stacking interaction between the TO base surrogate and the dsRNA part of the triplex. We expect that these results will provide a molecular basis for designing strong binding tFIT probes for fluorescence sensing of various kinds of purine-rich dsRNAs sequences including those carrying a pyrimidine-purine inversion. The obtained data also offers a new insight into further development of the universal bases incorporated in TFP. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A twice-as-smart synthetic G-quartet: PyroTASQ is both a smart quadruplex ligand and a smart fluorescent probe.

PubMed

Laguerre, Aurélien; Stefan, Loic; Larrouy, Manuel; Genest, David; Novotna, Jana; Pirrotta, Marc; Monchaud, David

2014-09-03

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Effective binding of perhalogenated closo-borates to serum albumins revealed by spectroscopic and ITC studies

NASA Astrophysics Data System (ADS)

Kuperman, Marina V.; Losytskyy, Mykhaylo Yu.; Bykov, Alexander Yu.; Yarmoluk, Sergiy M.; Zhizhin, Konstantin Yu.; Kuznetsov, Nikolay T.; Varzatskii, Oleg A.; Gumienna-Kontecka, Elzbieta; Kovalska, Vladyslava B.

2017-08-01

The interactions of boron cluster compounds closo-borates with biomolecules are widely studied due to their efficiency as agents for boron neutron capture therapy of cancer. In present work the binding abilities of anionic halogen closo-borates [B10Hal10]2- (Hal = Cl, Br, I) and [B12Hal12]2- (Hal = Cl, I) towards bovine and human serum albumins were investigated by spectroscopic and isothermal titration calorimetry (ITC) methods. The protein fluorescence quenching method and ITC studies confirmed the complex formation. The degree of protein fluorescence quenching increased from chlorine to iodine boron derivatives that is attributed to external heavy atom effect. The ITC data point on the existence in the protein structure of two types of binding sites: with higher and lower affinity to closo-borates. Albumin-closo-borate complex binding ratio, n (4-5 anions per protein molecule) is higher than for the parent hydrogen closo-borates (2 anions per protein molecule). Binding constants estimated by fluorescent and ITC methods indicate higher affinity of halogen closo-borates to albumins (K in the range of 104-106 M-1) comparing to that of the hydrogen closo-borate (K about 103 M-1). Due to their high affinity and high binding ratio to albumins halogen closo-borates are proposed for further studies as agents for boron neutron capture therapy.

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Synthesis strategy: building a culturally sensitive mid-range theory of risk perception using literary, quantitative, and qualitative methods.

PubMed

Siaki, Leilani A; Loescher, Lois J; Trego, Lori L

2013-03-01

This article presents a discussion of development of a mid-range theory of risk perception. Unhealthy behaviours contribute to the development of health inequalities worldwide. The link between perceived risk and successful health behaviour change is inconclusive, particularly in vulnerable populations. This may be attributed to inattention to culture. The synthesis strategy of theory building guided the process using three methods: (1) a systematic review of literature published between 2000-2011 targeting perceived risk in vulnerable populations; (2) qualitative and (3) quantitative data from a study of Samoan Pacific Islanders at high risk of cardiovascular disease and diabetes. Main concepts of this theory include risk attention, appraisal processes, cognition, and affect. Overarching these concepts is health-world view: cultural ways of knowing, beliefs, values, images, and ideas. This theory proposes the following: (1) risk attention varies based on knowledge of the health risk in the context of health-world views; (2) risk appraisals are influenced by affect, health-world views, cultural customs, and protocols that intersect with the health risk; (3) strength of cultural beliefs, values, and images (cultural identity) mediate risk attention and risk appraisal influencing the likelihood that persons will engage in health-promoting behaviours that may contradict cultural customs/protocols. Interventions guided by a culturally sensitive mid-range theory may improve behaviour-related health inequalities in vulnerable populations. The synthesis strategy is an intensive process for developing a culturally sensitive mid-range theory. Testing of the theory will ascertain its usefulness for reducing health inequalities in vulnerable groups. © 2012 Blackwell Publishing Ltd.

Direct magnitude estimates of speech intelligibility in dysarthria: effects of a chosen standard.

PubMed

Weismer, Gary; Laures, Jacqueline S

2002-06-01

Direct magnitude estimation (DME) has been used frequently as a perceptual scaling technique in studies of the speech intelligibility of persons with speech disorders. The technique is typically used with a standard, or reference stimulus, chosen as a good exemplar of "midrange" intelligibility. In several published studies, the standard has been chosen subjectively, usually on the basis of the expertise of the investigators. The current experiment demonstrates that a fixed set of sentence-level utterances, obtained from 4 individuals with dysarthria (2 with Parkinson disease, 2 with traumatic brain injury) as well as 3 neurologically normal speakers, is scaled differently depending on the identity of the standard. Four different standards were used in the main experiment, three of which were judged qualitatively in two independent evaluations to be good exemplars of midrange intelligibility. Acoustic analyses did not reveal obvious differences between these four standards but suggested that the standard with the worst-scaled intelligibility had much poorer voice source characteristics compared to the other three standards. Results are discussed in terms of possible standardization of midrange intelligibility exemplars for DME experiments.

Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

PubMed

Matthaeus, Friederike; Schloss, Patrick; Lau, Thorsten

2015-12-16

The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

NASA Astrophysics Data System (ADS)

Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

2009-02-01

Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

Biophysical and computational comparison on the binding affinity of three important nutrients to β-lactoglobulin: folic acid, ascorbic acid and vitamin K3.

PubMed

Shahraki, Somaye; Heydari, Ali; Saeidifar, Maryam; Gomroki, Masoumeh

2017-11-06

Small globular protein, β-lactoglobulin (βLG), which has significant affinity toward many drugs, is the most abundant whey protein in milk. In this study, the interaction of βLG with three important nutrients, ascorbic acid (ASC), folic acid (FOL), and vitamin K3 (VK3) was investigated by spectroscopic methods (UV-visible and fluorescence) along with molecular docking technique. The results of fluorescence measurements showed that studied nutrients strongly quenched βLG fluorescence in static (FOL and ACS) or static-dynamic combined quenching (VK3) mode. The values of binding constants (K βLG-ASC  ~ 4.34 × 10 4  M -1 , K βLG-FOL ~ 1.67 × 10 4  M -1 and K βLG-VK3 ~ 13.49 × 10 4  M -1 at 310 K) suggested that VK3 and FOL had stronger binding affinity toward βLG than ASC. Thermodynamic analysis indicated that hydrophobic interactions are the major forces in the stability of FOL-βLG complex with enthalpy- and entropy-driving mode while, hydrogen bonds and van der Waals interactions play a major role for βLG-ASC and βLG-VK3 associations. The results of 3D fluorescence FT-IR and UV-Visible measurements indicated that the binding of above nutrients to βLG may induce conformational and micro-environmental changes of protein. Also, there is a reciprocal complement between spectroscopic techniques and molecular docking modeling. The docking results indicate that the ASC, FOL, and VK3 bind to residues located in the subdomain B of βLG. Finally, this report suggests that βLG could be used as an effective carrier of above nutrients in functional foods.

Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe.

PubMed

Ren, Xiao M; Guo, Liang-Hong

2012-04-17

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein-thyroxine (F-T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F-T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F-T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F-T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F-T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F-T4 bound to the proteins at the T4 binding site. By using F-T4 as the fluorescence probe in competitive binding assays, 11 OH-PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH-BDE-47 and 3'-OH-BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules.

PubMed

Newton, Ana S; Deiana, Luca; Puleo, David E; Cisneros, José A; Cutrona, Kara J; Schlessinger, Joseph; Jorgensen, William L

2017-06-08

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as K d results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules

PubMed Central

2017-01-01

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses. PMID:28626520

Methods for quantifying T cell receptor binding affinities and thermodynamics

PubMed Central

Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

2013-01-01

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. PMID:21609868

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+.

PubMed

Li, Yan; Li, Lianzhi; Pu, Xuewei; Ma, Guolin; Wang, Erqiong; Kong, Jinming; Liu, Zhipeng; Liu, Yangzhong

2012-06-15

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Effect of phloretin on the binding of 1-anilino-8-naphtalene sulfonate (ANS) to 1,2-Dimyristoyl-sn-glycero-3-phosphocoline (DMPC) vesicles in the gel and liquid-crystalline state.

PubMed

Cutró, Andrea C; Montich, Guillermo; Roveri, Oscar A

2015-02-01

Phloretin is a known modifier of the internal dipole potential of lipid membranes. We studied the interaction of phloretin with model lipid membranes and how it influences the membrane dipole organization using ANS as fluorescent probe. The fluorescence increase observed when ANS binds to DMPC liposomes in gel phase (13 °C) was 2.5 times larger in the presence of phloretin. This effect was due to an increase in ANS affinity, which can be related to the known capability of phloretin in decreasing the dipole potential. Conversely, when the experiments were carried out at 33 °C (liquid crystalline phase), phloretin completely inhibited the increase in ANS fluorescence. In addition, phloretin only affected the electrical properties of the membrane in the gel phase, whereas it modifies structural ones in the liquid-crystalline state. We postulate that phloretin was bound only to the DMPC interface in the gel phase decreasing the surface negative charge density without modifying the structural properties of the ANS binding sites. In the liquid-crystalline phase instead, it increased the accessibility of water to the ANS binding sites decreasing the intrinsic affinity and the fluorescence quantum yield of ANS.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hall, Justin; Brault, Amy; Vincent, Fabien

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

Enantiomers of Single-Wall Carbon Nanotubes Show Distinct Coating Displacement Kinetics.

PubMed

Zheng, Yu; Bachilo, Sergei M; Weisman, R Bruce

2018-06-27

It is known that specific oligomers of single-stranded DNA (ssDNA) can show remarkable selectivity when coating different structural species of single-wall carbon nanotubes (SWCNTs). We report that (ATT) 4 ssDNA coatings strongly distinguish between the two optical isomers of (7,5) SWCNTs. This causes resolvable shifts in their fluorescence spectra and differences of 2 orders of magnitude in the room temperature rates of coating displacement, as monitored through changes in nanotube fluorescence wavelength and intensity on exposure to sodium deoxycholate. During coating displacement, the enantiomer with high affinity for the ssDNA oligomer is deduced to form an intermediate hybrid that is not observed for the low affinity enantiomer. These results reveal that enantiomeric differences in SWCNTs complexed with ssDNA are more diverse and dramatic than previously recognized.

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

PubMed

Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

2016-05-18

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

Investigation of the interaction between berberine and nucleosomes in solution: Spectroscopic and equilibrium dialysis approach

NASA Astrophysics Data System (ADS)

Rabbani-Chadegani, Azra; Mollaei, Hossein; Sargolzaei, Javad

2017-02-01

Berberine is a natural plant alkaloid with high pharmacological potential. Although its interaction with free DNA has been the subject of several reports, to date there is no work concerning the effect of berberine on nucleoprotein structure of DNA, the nucleosomes. The present study focuses on the binding affinity of berberine to nucleosomes and histone H1 employing various spectroscopic techniques, fluorescence, circular dichroism, thermal denaturation as well as equilibrium dialysis. The results showed that the binding of berberine to nucleosomes is positive cooperative with Ka = 5.57 × 103 M- 1. Berberine quenched with the chromophores of protein moiety of nucleosomes and reduced fluorescence emission intensity at 335 nm with Ksv value of 0.135. Binding of berberine to nucleosomes decreased the absorbance at 210 and 260 nm, produced hypochromicity in thermal denaturation profiles and its affinity to nucleoprotein structure of nucleosomes was much higher than to free DNA. Berberine also exhibited high affinity to histone H1 in solution and the binding was positive cooperative with. Ka = 3.61 × 103 M- 1. Moreover berberine decreased fluorescence emission intensity of H1 by quenching with tyrosine residue in its globular core domain. The circular dichroism profiles demonstrated that the binding of drug induced secondary structural changes in both DNA stacking and histone H1. It is concluded that berberine is genotoxic drug, interacts with nucleosomes and in this process histone H1 is involved to exert its anticancer activity.

Molecular Interaction Between Salivary Proteins and Food Tannins.

PubMed

Silva, Mafalda Santos; García-Estévez, Ignacio; Brandão, Elsa; Mateus, Nuno; de Freitas, Victor; Soares, Susana

2017-08-09

Polyphenols interaction with salivary proteins (SP) has been related with organoleptic features such as astringency. The aim of this work was to study the interaction between some human SP and tannins through two spectroscopic techniques, fluorescence quenching, and saturation transfer difference-nuclear magnetic resonance (STD-NMR). Generally, the results showed a significant interaction between SP and both condensed tannins and ellagitannins. Herein, STD-NMR proved to be a useful tool to map tannins' epitopes of binding, while fluorescence quenching allowed one to discriminate binding affinities. Ellagitannins showed the greatest binding constants values (K SV from 20.1 to 94.1 mM -1 ; K A from 0.7 to 8.3 mM -1 ) in comparison with procyanidins (K SV from 5.4 to 40.0 mM -1 ; K A from 1.1 to 2.7 mM -1 ). In fact, punicalagin was the tannin that demonstrated the highest affinity for all three SP. Regarding SP, P-B peptide was the one with higher affinity for ellagitannins. On the other hand, cystatins showed in general the lower K SV and K A values. In the case of condensed tannins, statherin was the SP with the highest affinity, contrasting with the other two SP. Altogether, these results are evidence that the distinct SP present in the oral cavity have different abilities to interact with food tannins class.

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

A label-free fluorescent probe for Hg2+ and biothiols based on graphene oxide and Ru-complex

PubMed Central

Wang, Linlin; Yao, Tianming; Shi, Shuo; Cao, Yanlin; Sun, Wenliang

2014-01-01

A novel, selective and sensitive switch-on fluorescent sensor for Hg2+ and switch-off fluorescent probe for biothiols was developed by using [Ru(bpy)2(pip)]2+ as the signal reporter and graphene oxide (GO) as the quencher. Due to the affinity of GO towards single-stranded DNA (ss-DNA) and [Ru(bpy)2(pip)]2+, the three components assembled, resulting in fluorescence quenching. Upon addition of Hg2+, a double-stranded DNA (ds-DNA) via T–Hg2+–T base pairs was formed, and [Ru(bpy)2(pip)]2+ intercalated into the newly formed ds-DNA. Then, [Ru(bpy)2(pip)]2+ and ds-DNA were removed from the surface of GO, resulting in the restoration of fluorescence. Subsequently, upon addition of biothiols, Hg2+ was released from ds-DNA, due to the higher affinity of Hg2+ to the sulfur atoms of biothiols, which could induce ds-DNA unwinding to form ss-DNA. Then ss-DNA and [Ru(bpy)2(pip)]2+ were adsorbed on the surface of GO, the fluorescence of [Ru(bpy)2(pip)]2+ was quenched again. Therefore, the changes in emission intensity of [Ru(bpy)2(pip)]2+ directly correlated to the amount of detection target (Hg2+ or biothiols) in solution. The assay exhibited high sensitivity and selectivity, with the limits of detection for Hg2+, cysteine (Cys) and glutathione (GSH) to be 2.34 nM, 6.20 nM and 4.60 nM, respectively. PMID:24936798

Subject-Specific Carpal Ligament Elongation in Extreme Positions, Grip, and the Dart Thrower's Motion

PubMed Central

Rainbow, Michael J.; Kamal, Robin N.; Moore, Douglas C.; Akelman, Edward; Wolfe, Scott W.; Crisco, Joseph J.

2015-01-01

This study examined whether the radiocarpal and dorsal capsular ligaments limit end-range wrist motion or remain strained during midrange wrist motion. Fibers of these ligaments were modeled in the wrists of 12 subjects over multiple wrist positions that reflect high demand tasks and the dart thrower's motion. We found that many of the volar and dorsal ligaments were within 5% of their maximum length throughout the range of wrist motion. Our finding of wrist ligament recruitment during midrange and end-range wrist motion helps to explain the complex but remarkably similar intersubject patterns of carpal motion. PMID:26367853

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

Selective and Sensitive Fluorescent Detection of Picric Acid by New Pyrene and Anthracene Based Copper Complexes.

PubMed

Reddy, Kumbam Lingeshwar; Kumar, Anabathula Manoj; Dhir, Abhimanew; Krishnan, Venkata

2016-11-01

New pyrene and anthracene based copper complexes 4 and 7 respectively were designed, synthesized and characterized. The fluorescence behaviour of both 4 and 7 were evaluated towards nitro aromatics and anions. Both 4 and 7 possess high selectivity for the detection of well-known explosive picric acid (PA) by showing maximum fluorescence affinity. Furthermore, complex 4 showed similar sensing efficiency towards PA at different pH ranges. It was also used for real world applications, as illustrated by the very fast detection of PA from soil samples observed directly by naked eye.

Fluorescein-labeled stable neurotensin derivatives.

PubMed

Maes, Veronique; Hultsch, Christina; Kohl, Suzann; Bergmann, Ralf; Hanke, Thomas; Tourwé, Dirk

2006-08-01

Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

PubMed Central

Neumüller, Klaus G.; Elsayad, Kareem; Reisecker, Johannes M.; Waxham, M. Neal; Heinze, Katrin G.

2010-01-01

Background Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. Methodology/Principal Findings We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. Conclusions/Significance The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner. PMID:21124984

A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates

NASA Astrophysics Data System (ADS)

Rajasekhar, K.; Narayanaswamy, Nagarjun; Murugan, N. Arul; Kuang, Guanglin; Ågren, Hans; Govindaraju, T.

2016-04-01

A major challenge in the Alzheimer’s disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 107 M-1) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies.

In vivo fluorescence imaging of hepatocellular carcinoma using a novel GPC3-specific aptamer probe

PubMed Central

Zhao, Menglong; Dong, Lili; Liu, Zhuang; Yang, Shuohui

2018-01-01

Background Glypican-3 (GPC3) is highly expressed in most of the hepatocellular carcinomas (HCCs), even in small HCCs. It may be used as a potential biomarker for early detection of HCC. The aptamer is a promising targeting agent with unique advantages over antibody. This study was to introduce a novel GPC3 specific aptamer (AP613-1), to verify its specific binding property in vitro, and to evaluate its targeting efficiency in vivo by performing near-infrared (NIR) fluorescence imaging on an HCC xenograft model. Methods AP613-1 was generated from the systematic evolution of ligands by exponential enrichment. Flow cytometry and aptamer-based immunofluorescence imaging were performed to verify the binding affinity of AP613-1 to GPC3 in vitro. NIR Fluorescence images of nude mice with unilateral (n=12) and bilateral (n=4) subcutaneous xenograft tumors were obtained. Correlation between the tumor fluorescence intensities in vivo and ex vivo was analyzed. Results AP613-1 could specifically bind to GPC3 in vitro. In vivo and ex vivo tumors, fluorescence intensities were in excellent correlation (P<0.001, r=0.968). The fluorescence intensity is significantly higher in tumors given Alexa Fluor 750 (AF750) labeled AP613-1 than in those given AF750 labeled initial ssDNA library both in vivo (P<0.001) and ex vivo (P=0.022). In the mice with bilateral subcutaneous tumors injected with AF750 labeled AP613-1, Huh-7 tumors showed significantly higher fluorescence intensities than A549 tumors both in vivo (P=0.016) and ex vivo (P=0.004). Conclusions AP613-1 displays a specific binding affinity to GPC3 positive HCC. Fluorescently labeled AP613-1 could be used as an imaging probe to subcutaneous HCC in xenograft models. PMID:29675356

Investigation of the Binding Profiles of AZD2184 and Thioflavin T with Amyloid-β(1-42) Fibril by Molecular Docking and Molecular Dynamics Methods.

PubMed

Kuang, Guanglin; Murugan, N Arul; Tu, Yaoquan; Nordberg, Agneta; Ågren, Hans

2015-09-03

Detecting deposits of amyloid β fibrils in the brain is of paramount importance for an early diagnosis of Alzheimer's disease. A number of PET tracers have been developed for amyloid imaging, but many suffer from poor specificity and large signal to background ratio. Design of tracers with specificity and improved binding affinity requires knowledge about various potential binding sites in the amyloid β fibril available for the tracers and the nature of the local microenvironment of these sites. In this study we investigate the local structure of fibrils using two important probes, namely, thioflavin T (a fluorescent probe) and AZD2184 (a PET tracer). The target structures for amyloid-β(1-42) fibril are based on reported NMR solution models. By explicitly considering the effect of fibril flexibility on the available binding sites for all these models, the binding affinity of these probes has been investigated. The binding profiles of AZD2184 and thioflavin T were studied by molecular docking and molecular dynamics simulation methods. The two compounds were found to bind at the same sites of the fibril: three of which are within the fibril, and one is on the two sides of the Met35 residue on the surface. The binding affinity of AZD2184 and thioflavin T is found to be higher at the core sites than on the surface due to more contact residues. The binding affinity of AZD2184 is much higher than that of thioflavin T at every site due to electrostatic interaction and spatial restriction, which is in good agreement with experimental observation. However, the structural change of thioflavin T is much more significant than that of AZD2184, which is the chemical basis for its usage as a fluorescent probe. The ramifications of these results for the design and optimization of PET radioligands and fluorescent probes are briefly discussed.

Selection and characterization of a DNA aptamer to crystal violet.

PubMed

Chen, Yang; Wang, Jine; Zhang, Yajie; Xu, Lijun; Gao, Tian; Wang, Bing; Pei, Renjun

2018-06-13

Aptamers are short single-stranded DNA or RNA, which can be selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). In order to develop novel light-up probes to substitute G-quadruplex (G4), we selected a DNA aptamer for crystal violet (CV), a triphenylmethane light-up dye, by a modified affinity chromatography-based SELEX. The ssDNA pool was first coupled on streptavidin-coated agarose beads through a biotin labeled complementary oligonucleotide, and then the aptamer sequences would be released from agarose beads by CV affinity. This method is simple, straightforward and effective. The aptamer sequence with a low micromolar dissociation constant (Kd) and good specificity was achieved after 11 rounds of selection. The light-up properties of the CV-aptamer were also investigated, and the CV showed dramatic fluorescence enhancement. The CV-aptamer pair could be further used as a novel light-up fluorescent probe to design biosensors.

Carbodiimide versus click chemistry for nanoparticle surface functionalization: a comparative study for the elaboration of multimodal superparamagnetic nanoparticles targeting αvβ3 integrins.

PubMed

Bolley, Julie; Guenin, Erwann; Lievre, Nicole; Lecouvey, Marc; Soussan, Michael; Lalatonne, Yoann; Motte, Laurence

2013-11-26

Superparamagnetic fluorescent nanoparticles targeting αvβ3 integrins were elaborated using two methodologies: carbodiimide coupling and click chemistries (CuACC and thiol-yne). The nanoparticles are first functionalized with hydroxymethylenebisphonates (HMBP) bearing carboxylic acid or alkyne functions. Then, a large number of these reactives functions were used for the covalent coupling of dyes, poly(ethylene glycol) (PEG), and cyclic RGD. Several methods were used to characterize the nanoparticle surface functionalization, and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI. The affinity toward integrins was evidenced by solid-phase receptor-binding assay. In addition to their chemoselective natures, click reactions were shown to be far more efficient than the carbodiimide coupling. The grafting increase was shown to enhance targeting affinity to integrin without imparing MRI and fluorescent properties.

Insights into the interaction of methotrexate and human serum albumin: A spectroscopic and molecular modeling approach.

PubMed

Cheng, Li-Yang; Fang, Min; Bai, Ai-Min; Ouyang, Yu; Hu, Yan-Jun

2017-08-01

In this study, fluorescence spectroscopy and molecular modeling approaches were employed to investigate the binding of methotrexate to human serum albumin (HSA) under physiological conditions. From the mechanism, it was demonstrated that fluorescence quenching of HSA by methotrexate results from the formation of a methotrexate/HSA complex. Binding parameters calculated using the Stern-Volmer method and the Scatchard method showed that methotrexate binds to HSA with binding affinities in the order 10 4  L·mol -1 . Thermodynamic parameter studies revealed that the binding reaction is spontaneous, and that hydrogen bonds and van der Waals interactions play a major role in the reaction. Site marker competitive displacement experiments and a molecular modeling approach demonstrated that methotrexate binds with appropriate affinity to site I (subdomain IIA) of HSA. Furthermore, we discuss some factors that influence methotrexate binding to HSA. Copyright © 2017 John Wiley & Sons, Ltd.

On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies.

PubMed

Tahtaoui, Chouaib; Guillier, Fabrice; Klotz, Philippe; Galzi, Jean-Luc; Hibert, Marcel; Ilien, Brigitte

2005-12-01

The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.

A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH

NASA Astrophysics Data System (ADS)

Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

2015-06-01

Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (Ka = 3582.88 M-1) and selectivity for fructose over glucose at pH = 7.4. The sensor 1 showed a linear response toward D-fructose in the concentrations ranging from 2.5 × 10-5 to 4 × 10-4 mol L-1 with the detection limit of 1.3 × 10-5 mol L-1.

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Molecular interaction of 2,4-diacetylphloroglucinol (DAPG) with human serum albumin (HSA): The spectroscopic, calorimetric and computational investigation

NASA Astrophysics Data System (ADS)

Pragna Lakshmi, T.; Mondal, Moumita; Ramadas, Krishna; Natarajan, Sakthivel

2017-08-01

Drug molecule interaction with human serum albumin (HSA) affects the distribution and elimination of the drug. The compound, 2,4-diacetylphloroglucinol (DAPG) has been known for its antimicrobial, antiviral, antihelminthic and anticancer properties. However, its interaction with HSA is not yet reported. In this study, the interaction between HSA and DAPG was investigated through steady-state fluorescence, time-resolved fluorescence (TRF), circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, isothermal titration calorimetry (ITC), molecular docking and molecular dynamics simulation (MDS). Fluorescence spectroscopy results showed the strong quenching of intrinsic fluorescence of HSA due to interaction with DAPG, through dynamic quenching mechanism. The compound bound to HSA with reversible and moderate affinity which explained its easy diffusion from circulatory system to target tissue. The thermodynamic parameters from fluorescence spectroscopic data clearly revealed the contribution of hydrophobic forces but, the role of hydrogen bonds was not negligible according to the ITC studies. The interaction was exothermic and spontaneous in nature. Binding with DAPG reduced the helical content of protein suggesting the unfolding of HSA. Site marker fluorescence experiments revealed the change in binding constant of DAPG in the presence of site I (warfarin) but not site II marker (ibuprofen) which confirmed that the DAPG bound to site I. ITC experiments also supported this as site I marker could not bind to HSA-DAPG complex while site II marker was accommodated in the complex. In silico studies further showed the lowest binding affinity and more stability of DAPG in site I than in site II. Thus the data presented in this study confirms the binding of DAPG to the site I of HSA which may help in further understanding of pharmacokinetic properties of DAPG.

Structural locus of transmucosal albumin efflux in canine ileum. A fluorescent study.

PubMed

Granger, D N; Cook, B H; Taylor, A E

1976-12-01

This study demonstrates the effects of elevated intestinal venous pressure on the intestinal tissue spaces and the histological locus of the transmucosal albumin flux under such conditions. The authors were able to localize albumin in the tissues using an Evans blue-albumin fluorescence technique. This technique makes use of the fluorescence properties and albumin affinity of Evans blue dye (T-1824). Evans blue dye has a high affinity for albumin and emits a red-orange fluorescence at a wavelength of 720 nm. Evans blue was mixed with a solution of bovine serum albumin at concentrations that yield negligible amounts of free dye. Control ileal samples were obtained in order to visualize the natural tissue morphology and fluorescence. The Evans blue-albumin solution was injected and tissue samples were obtained 15 and 60 min postinjection, then venous outflow was occluded and after 15 and 60 min the tissues were sampled. Each sample was immediately frozen, freeze dried, embedded in paraffin, and 7-mu sections were made. The Evans blue-albumin was demonstrated histologically with a fluorescence microscope. No leakage sites were apparent at normal venous pressures. However, after elevation of venous pressure, Evans blue-albumin was observed in the interepithelial and/or intraepithelial spaces of villus tips, but no Evans blue-albumin was observed either between or within the epithelial cells of the crypts, or within the tubular crypt lumina. These results indicate that at elevated venous pressures, the transmucosal albumin flux occurs exclusively at the villus tip region, suggesting a great vulnerability of the cells found in this region to elevations in tissue pressure as compared to the crypt epithelial cells.

Interaction of amphotericin B and its low toxic derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method.

PubMed

Szlinder-Richert, Joanna; Cybulska, Barbara; Grzybowska, Jolanta; Bolard, Jacques; Borowski, Edward

2004-04-01

Amphotericin B (AMB) derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Yijia; Ford, Nicole R.; Hecht, Karen A.

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv ormore » the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.« less

Comprehensive study of interaction between biocompatible PEG-InP/ZnS QDs and bovine serum albumin.

PubMed

Sannaikar, M S; Inamdar, Laxmi S; Pujar, G H; Wari, M N; Balasinor, Nafisa H; Inamdar, S R

2018-05-01

Polyethylene glycol (PEG) surface modified biocompatible InP/ZnS quantum dots (QDs) act as a potential alternative for conventional carcinogenic cadmium-based quantum dots for in vivo and in vitro studies. Comprehensively, we studied the interaction between a model protein bovine serum albumin (BSA) and PEGylated toxic free InP/ZnS QDs using various spectroscopic tools such as absorption, fluorescence quenching, time resolved and synchronous fluorescence spectroscopic measurements. These studies principally show that tryptophan (Trp) residues of BSA have preferable binding affinity towards PEG-InP/ZnS QDs surface and a blue shift in Trp fluorescence emission is a signature of conformational changes in its hydrophobic microenvironment. Photoluminescence (PL) intensity of Trp is quenched by ground state complex formation (static quenching) at room temperature. However, InP/ZnS@BSA conjugates become unstable with increasing temperature and PL intensity of Trp is quenched via dynamic quenching by PEG-InP/ZnS QDs. Experimentally determined thermodynamic parameters for these conjugates have shown spontaneity, entropy driven and exothermic nature of bio-conjugation. The calculated binding affinity (n ≅ 1, Hill coefficient) suggest that the affinity of InP/ZnS QDs for a BSA protein is not dependent on whether or not other BSA proteins are already bound to the QD surface. Energy transfer efficiency (E), Trp residue to InP/ZnS QDs distances and energy transfer rate (k T ) were all obtained from FÖrster resonance energy. Copyright © 2017 John Wiley & Sons, Ltd.

Fluorescence and computational studies of thymidine phosphorylase affinity toward lipidated 5-FU derivatives

NASA Astrophysics Data System (ADS)

Lettieri, R.; D'Abramo, M.; Stella, L.; La Bella, A.; Leonelli, F.; Giansanti, L.; Venanzi, M.; Gatto, E.

2018-04-01

Thymidine phosphorylase (TP) is an enzyme that is up-regulated in a wide variety of solid tumors, including breast and colorectal cancers. It is involved in tumor growth and metastasis, for this reason it is one of the key enzyme to be inhibited, in an attempt to prevent tumor proliferation. However, it also plays an active role in cancer treatment, through its contribution in the conversion of the anti-cancer drug 5-fluorouracil (5-FU) to an irreversible inhibitor of thymidylate synthase (TS), responsible of the inhibition of the DNA synthesis. In this work, the intrinsic TP fluorescence has been investigated for the first time and exploited to study TP binding affinity for the unsubstituted 5-FU and for two 5-FU derivatives, designed to expose this molecule on liposomal membranes. These molecules were obtained by functionalizing the nitrogen atom with a chain consisting of six (1) or seven (2) units of glycol, linked to an alkyl moiety of 12 carbon atoms. Derivatives (1) and (2) exhibited an affinity for TP in the micromolar range, 10 times higher than the parent compound, irrespective of the length of the polyoxyethylenic spacer. This high affinity was maintained also when the compounds were anchored in liposomal membranes. Experimental results were supported by molecular dynamics simulations and docking calculations, supporting a feasible application of the designed supramolecular lipid structure in selective targeting of TP, to be potentially used as a drug delivery system or sensor device.

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

PubMed

Mainali, Dipak; Smith, Emily A

2013-04-01

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

PubMed Central

Adam, Yoav; Tayer, Naama; Rotem, Dvir; Schreiber, Gideon; Schuldiner, Shimon

2007-01-01

EmrE is an Escherichia coli H+-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP+, a high-affinity substrate, is 2 × 107 M−1·s−1, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the koff of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs. PMID:17984053

The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

NASA Astrophysics Data System (ADS)

Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

2016-02-01

The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

PubMed Central

Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-01

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

PubMed

Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-15

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.

PubMed

Pragl, Bernt; Koschak, Alexandra; Trieb, Maria; Obermair, Gerald; Kaufmann, Walter A; Gerster, Uli; Blanc, Eric; Hahn, Christoph; Prinz, Heino; Schütz, Gerhard; Darbon, Herve; Gruber, Hermann J; Knaus, Hans-Günther

2002-01-01

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

Boronic acid recognition of non-interacting carbohydrates for biomedical applications: increasing fluorescence signals of minimally interacting aldoses and sucralose.

PubMed

Resendez, Angel; Halim, Md Abdul; Singh, Jasmeet; Webb, Dominic-Luc; Singaram, Bakthan

2017-11-22

To address carbohydrates that are commonly used in biomedical applications with low binding affinities for boronic acid based detection systems, two chemical modification methods were utilized to increase sensitivity. Modified carbohydrates were analyzed using a two component fluorescent probe based on boronic acid-appended viologen-HPTS (4,4'-o-BBV). Carbohydrates normally giving poor signals (fucose, l-rhamnose, xylose) were subjected to sodium borohydride (NaBH 4 ) reduction in ambient conditions for 1 h yielding the corresponding sugar alcohols from fucose, l-rhamnose and xylose in essentially quantitative yields. Compared to original aldoses, apparent binding affinities were increased 4-25-fold. The chlorinated sweetener and colon permeability marker sucralose (Splenda), otherwise undetectable by boronic acids, was dechlorinated to a detectable derivative by reactive oxygen and hydroxide intermediates by the Fenton reaction or by H 2 O 2 and UV light. This method is specific to sucralose as other common sugars, such as sucrose, do not contain any carbon-chlorine bonds. Significant fluorescence response was obtained for chemically modified sucralose with the 4,4'-o-BBV-HPTS probe system. This proof of principle can be applied to biomedical applications, such as gut permeability, malabsorption, etc.

Accounting for climate and air quality damages in future U.S. electricity generation scenarios.

PubMed

Brown, Kristen E; Henze, Daven K; Milford, Jana B

2013-04-02

The EPA-MARKAL model of the U.S. electricity sector is used to examine how imposing emissions fees based on estimated health and environmental damages might change electricity generation. Fees are imposed on life-cycle emissions of SO(2), nitrogen oxides (NO(x)), particulate matter, and greenhouse gases (GHG) from 2015 through 2055. Changes in electricity production, fuel type, emissions controls, and emissions produced under various fees are examined. A shift in fuels used for electricity production results from $30/ton CO(2)-equivalent GHG fees or from criteria pollutant fees set at the higher-end of the range of published damage estimates, but not from criteria pollutant fees based on low or midrange damage estimates. With midrange criteria pollutant fees assessed, SO(2) and NOx emissions are lower than the business as usual case (by 52% and 10%, respectively), with larger differences in the western U.S. than in the eastern U.S. GHG emissions are not significantly impacted by midrange criteria pollutant fees alone; conversely, with only GHG fees, NO(x) emissions are reduced by up to 11%, yet SO(2) emissions are slightly higher than in the business as usual case. Therefore, fees on both GHG and criteria pollutants may be needed to achieve significant reductions in both sets of pollutants.

Enhanced fluorescence of tetrasulfonated zinc phthalocyanine by graphene quantum dots and its application in molecular sensing/imaging.

PubMed

Wang, Jian; Zhang, Yanjun; Ye, Jiqing; Jiang, Zhou

2017-06-01

When excited at 435 nm, tetra-sulfonate zinc phthalocyanine (ZnPcS 4 ) emitted dual fluorescence at 495 and 702 nm. The abnormal fluorescence at 495 nm was experimentally studied and analyzed in detail for the first time. The abnormal fluorescence at 495 nm was deduced to originate from triplet-triplet (T-T) energy transfer of excited phthalocyanine ( 3 *ZnPcS 4 ). Furthermore, graphene quantum dots (GQDs) enhanced the 495 nm fluorescence quantum yield (Q) of ZnPcS 4 . The fluorescence properties of ZnPcS 4 -GQDs conjugate were retained in a cellular environment. Based on the fluorescence of ZnPcS 4 -GQDs conjugate, we designed and prepared an Apt29/thrombin/Apt15 sandwich thrombin sensor with high specificity and affinity. This cost-saving, simple operational sensing strategy can be extended to use in sensing/imaging of other biomolecules. Copyright © 2016 John Wiley & Sons, Ltd.

Semiconductor Nanomaterials-Based Fluorescence Spectroscopic and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometric Approaches to Proteome Analysis

PubMed Central

Kailasa, Suresh Kumar; Cheng, Kuang-Hung; Wu, Hui-Fen

2013-01-01

Semiconductor quantum dots (QDs) or nanoparticles (NPs) exhibit very unusual physico-chemcial and optical properties. This review article introduces the applications of semiconductor nanomaterials (NMs) in fluorescence spectroscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for biomolecule analysis. Due to their unique physico-chemical and optical properties, semiconductors NMs have created many new platforms for investigating biomolecular structures and information in modern biology. These semiconductor NMs served as effective fluorescent probes for sensing proteins and cells and acted as affinity or concentrating probes for enriching peptides, proteins and bacteria proteins prior to MALDI-MS analysis. PMID:28788422

Fluorescence Imaging/Agents in Tumor Resection.

PubMed

Stummer, Walter; Suero Molina, Eric

2017-10-01

Intraoperative fluorescence imaging allows real-time identification of diseased tissue during surgery without being influenced by brain shift and surgery interruption. 5-Aminolevulinic acid, useful for malignant gliomas and other tumors, is the most broadly explored compound approved for fluorescence-guided resection. Intravenous fluorescein sodium has recently received attention, highlighting tumor tissue based on extravasation at the blood-brain barrier (defective in many brain tumors). Fluorescein in perfused brain, unselective extravasation in brain perturbed by surgery, and propagation with edema are concerns. Fluorescein is not approved but targeted fluorochromes with affinity to brain tumor cells, in development, may offer future advantages. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Spectroscopic and molecular modeling study on the separate and simultaneous bindings of alprazolam and fluoxetine hydrochloride to human serum albumin (HSA): With the aim of the drug interactions probing

NASA Astrophysics Data System (ADS)

Dangkoob, Faeze; Housaindokht, Mohmmad Reza; Asoodeh, Ahmad; Rajabi, Omid; Rouhbakhsh Zaeri, Zeinab; Verdian Doghaei, Asma

2015-02-01

The objective of the present research is to study the interaction of separate and simultaneous of alprazolam (ALP) and fluoxetine hydrochloride (FLX) with human serum albumin (HSA) in phosphate buffer (pH 7.4) using different kinds of spectroscopic, cyclic voltammetry and molecular modeling techniques. The absorbance spectra of protein, drugs and protein-drug showed complex formation between the drugs and HSA. Fluorescence analysis demonstrated that ALP and FLX could quench the fluorescence spectrum of HSA and demonstrated the conformational change of HSA in the presence of both drugs. Also, fluorescence quenching mechanism of HSA-drug complexes both separately and simultaneously was suggested as static quenching. The analysis of UV absorption data and the fluorescence quenching of HSA in the binary and ternary systems showed that FLX decreased the binding affinity between ALP and HSA. On the contrary, ALP increased the binding affinity of FLX and HSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra indicated that the binding of drugs to HSA would modify the microenvironment around the Trp and Tyr residues and the conformation of HSA. The distances between Trp residue and the binding sites of the drugs were estimated according to the Förster theory, and it was demonstrated that non-radiative energy transfer from HSA to the drugs occurred with a high probability. Moreover, according to CV measurements, the decrease of peak current in the cyclic voltammogram of the both drugs in the presence of HSA revealed that they interacted with albumin and binding constants were calculated for binary systems which were in agreement with the binding constants obtained from UV absorption and fluorescence spectroscopy. The prediction of the best binding sites of ALP and FLX in binary and ternary systems in molecular modeling approach was done using of Gibbs free energy.

Spectroscopic and molecular modeling study on the separate and simultaneous bindings of alprazolam and fluoxetine hydrochloride to human serum albumin (HSA): with the aim of the drug interactions probing.

PubMed

Dangkoob, Faeze; Housaindokht, Mohmmad Reza; Asoodeh, Ahmad; Rajabi, Omid; Rouhbakhsh Zaeri, Zeinab; Verdian Doghaei, Asma

2015-02-25

The objective of the present research is to study the interaction of separate and simultaneous of alprazolam (ALP) and fluoxetine hydrochloride (FLX) with human serum albumin (HSA) in phosphate buffer (pH 7.4) using different kinds of spectroscopic, cyclic voltammetry and molecular modeling techniques. The absorbance spectra of protein, drugs and protein-drug showed complex formation between the drugs and HSA. Fluorescence analysis demonstrated that ALP and FLX could quench the fluorescence spectrum of HSA and demonstrated the conformational change of HSA in the presence of both drugs. Also, fluorescence quenching mechanism of HSA-drug complexes both separately and simultaneously was suggested as static quenching. The analysis of UV absorption data and the fluorescence quenching of HSA in the binary and ternary systems showed that FLX decreased the binding affinity between ALP and HSA. On the contrary, ALP increased the binding affinity of FLX and HSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra indicated that the binding of drugs to HSA would modify the microenvironment around the Trp and Tyr residues and the conformation of HSA. The distances between Trp residue and the binding sites of the drugs were estimated according to the Förster theory, and it was demonstrated that non-radiative energy transfer from HSA to the drugs occurred with a high probability. Moreover, according to CV measurements, the decrease of peak current in the cyclic voltammogram of the both drugs in the presence of HSA revealed that they interacted with albumin and binding constants were calculated for binary systems which were in agreement with the binding constants obtained from UV absorption and fluorescence spectroscopy. The prediction of the best binding sites of ALP and FLX in binary and ternary systems in molecular modeling approach was done using of Gibbs free energy. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel heterocyclic thiosemicarbazones derivatives as colorimetric and "turn on" fluorescent sensors for fluoride anion sensing employing hydrogen bonding.

PubMed

Ashok Kumar, S L; Saravana Kumar, M; Sreeja, P B; Sreekanth, A

2013-09-01

Two novel heterocyclic thiosemicarbazone derivatives have been synthesized, and characterized, by means of spectroscopic and single crystal X-ray diffraction methods. Their chromophoric-fluorogenic response towards anions in competing solvent dimethyl sulfoxide (DMSO) was studied. The receptor shows selective recognition towards fluoride anion. The binding affinity of the receptors with fluoride anion was calculated using UV-visible and fluorescence spectroscopic techniques. Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

The Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus†

PubMed Central

Quinlan, R. Jason; Reinhart, Gregory D.

2008-01-01

Differences between the crystal structures of inhibitor-bound and uninihibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits containing two different extrinsic fluorophores simultaneously in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel Fluorescence Correlation Spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor-binding, as binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated. PMID:16981693

Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase.

PubMed

Brewer, J M

1976-12-11

Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.

Interaction of Chelerythrine with Keyhole Limpet Hemocyanin: a Fluorescence Spectroscopy and Molecular Docking Study

NASA Astrophysics Data System (ADS)

Zhong, M.; Long, R. Q.; Wang, Y. H.; Chen, C. L.

2018-05-01

The quenching mechanism between chelerythrine (CHE) and keyhole limpet hemocyanin (KLH) was investigated using fluorescence spectroscopy and molecular docking. The experiments were conducted at three different temperatures (293, 298, and 303 K). The results revealed that the intrinsic fluorescence of KLH was strongly quenched by CHE through a static quenching mechanism. The thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction were calculated, indicating that the interaction between CHE and KLH was spontaneous and that van der Waals forces and hydrogen bond formation played major roles in the binding process. The intrinsic fluorescence of the tyrosine and tryptophan residues in KLH was studied by synchronous fluorescence, which suggested that CHE changed the conformation of KLH. Finally, molecular docking was used to obtain detailed information on the binding sites and binding affinities between CHE and KLH.

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

Ratiometric and turn-on monitoring for heavy and transition metal ions in aqueous solution with a fluorescent peptide sensor.

PubMed

Joshi, Bishnu Prasad; Park, Junwon; Lee, Wan In; Lee, Keun-Hyeung

2009-05-15

A novel fluorescent peptide sensor containing tryptophan (donor) and dansyl fluorophore (acceptor) was synthesized for monitoring heavy and transition metal (HTM) ions on the basis of metal ion binding motif (Cys-X-X-X-Cys). The peptide probe successfully exhibited a turn on and ratiometric response for several heavy metal ions such as Hg(2+), Cd(2+), Pb(2+), Zn(2+), and Ag(+) in aqueous solution. The enhancements of emission intensity were achieved in the presence of the HTM ions by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The detection limits of the sensor for Cd(2+), Pb(2+), Zn(2+), and Ag(+) were lower than the EPA's drinking water maximum contaminant levels (MCL). We described the fluorescent enhancement, binding affinity, and detection limit of the peptide probe for HTM ions.

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

PubMed

Yang, Rui-Nan; Li, Dong-Zhen; Yu, Guangqiang; Yi, Shan-Cheng; Zhang, Yinan; Kong, De-Xin; Wang, Man-Qun

2017-12-01

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

PubMed Central

García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

2015-01-01

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

β-Lactam Antibiotics with a High Affinity for PBP2 Act Synergistically with the FtsZ-Targeting Agent TXA707 against Methicillin-Resistant Staphylococcus aureus

PubMed Central

Ferrer-González, Edgar; Kaul, Malvika; Parhi, Ajit K.; LaVoie, Edmond J.

2017-01-01

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. We have developed a promising new FtsZ-targeting agent (TXA707) with potent activity against MRSA isolates resistant to current standard-of-care antibiotics. We present here results that demonstrate differing extents of synergy between TXA707 and a broad range of β-lactam antibiotics (including six cephalosporins, two penicillins, and two carbapenems) against MRSA. To explore whether there is a correlation between the extent of synergy and the preferential antibacterial target of each β-lactam, we determined the binding affinities of the β-lactam antibiotics for each of the four native penicillin-binding proteins (PBPs) of S. aureus using a fluorescence anisotropy competition assay. A comparison of the resulting PBP binding affinities with our corresponding synergy results reveals that β-lactams with a high affinity for PBP2 afford the greatest degree of synergy with TXA707 against MRSA. In addition, we present fluorescence and electron microscopy studies that suggest a potential mechanism underlying the synergy between TXA707 and the β-lactam antibiotics. In this connection, our microscopy results show a disruption of septum formation in TXA707-treated MRSA cells, with a concomitant mislocalization of the PBPs from midcell to nonproductive peripheral sites. Viewed as a whole, our results indicate that PBP2-targeting β-lactam antibiotics are optimal synergistic partners with FtsZ-targeting agents for use in combination therapy of MRSA infections. PMID:28630190

Improved Tumor Targeting and Longer Retention Time of NIR Fluorescent Probes Using Bioorthogonal Chemistry.

PubMed

Zhang, Xianghan; Wang, Bo; Zhao, Na; Tian, Zuhong; Dai, Yunpeng; Nie, Yongzhan; Tian, Jie; Wang, Zhongliang; Chen, Xiaoyuan

2017-01-01

The traditional labeling method for targeted NIR fluorescence probes requires directly covalent-bonded conjugation of targeting domains and fluorophores in vitro . Although this strategy works well, it is not sufficient for detecting or treating cancers in vivo , due to steric hindrance effects that relatively large fluorophore molecules exert on the configurations and physiological functions of specific targeting domains. The copper-free, "click-chemistry"-assisted assembly of small molecules in living systems may enhance tumor accumulation of fluorescence probes by improving the binding affinities of the targeting factors. Here, we employed a vascular homing peptide, GEBP11, as a targeting factor for gastric tumors, and we demonstrate its effectiveness for in vivo imaging via click-chemistry-mediated conjugation with fluorescence molecules in tumor xenograft mouse models. This strategy showed higher binding affinities than those of the traditional conjugation method, and our results showed that the tumor accumulation of click-chemistry-mediated probes are 11-fold higher than that of directly labeled probes. The tracking life was prolonged by 12-fold, and uptake of the probes into the kidney was reduced by 6.5-fold. For lesion tumors of different sizes, click-chemistry-mediated probes can achieve sufficient signal-to-background ratios (3.5-5) for in vivo detection, and with diagnostic sensitivity approximately 3.5 times that of traditional labeling probes. The click-chemistry-assisted detection strategy utilizes the advantages of "small molecule" probes while not perturbing their physiological functions; this enables tumor detection with high sensitivity and specific selectivity.

[Stimulation of DNA molecules association with amphiphilic derivatives of 1,3-diazaadamantane containing hydrophobic side chanins].

PubMed

Mamaeva, O K; Gabrielian, A G; Arutiunian, G L; Bocharova, T N; Smirnova, E A; Volodin, A A; Shchelkina, A K; Kaliuzhnyĭ, D N

2014-01-01

Earlier, a new class of compounds--amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides was revealed. Longer hydrophobic side chains of 1,3-diazaadamantanes promoted stronger acceleration of the reaction. In this study, interaction with DNA of two 1,3-diazaadamantane derivatives containing different side chains was investigated by use of optical methods. Concentration of the investigated 1,3-diazaadamantans micelles formation were determined by the means of monitoring fluorescence intensity enhancement of 1-anilinonaphtalene-8-sulphonate probe; as well as the ranges of concentrations where the compounds/water mixtures existed as true solutions. 1,3-diazaadamantanes affinity to DNA was determined with Fluorescent Intercalator Displacement (FID) approach. Significant increase in hydrodynamic volume of short DNA hairpins in the complexes with 1,3-diazaadamantanes was revealed by estimation of the fluorescence polarization of ethidium bromide probe bound to the hairpins. Intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantans was confirmed by Förster resonance energy transfer in system of an equimolar mixture of fluorescently labeled with Cy-3 and Cy-5 hairpins. In this study, the number of positive charges at 1,3-diazaadamantane derivatives containing side chains of different lengths was demonstrated to affect their affinity to DNA, whereas longer length of the hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate, in particular, DNA strand exchange.

A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH.

PubMed

Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

2015-06-05

Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (K(a)=3582.88 M(-1)) and selectivity for fructose over glucose at pH=7.4. The sensor 1 showed a linear response toward d-fructose in the concentrations ranging from 2.5×10(-5) to 4×10(-4) mol L(-1) with the detection limit of 1.3×10(-5) mol L(-1). Copyright © 2015 Elsevier B.V. All rights reserved.

Malathion-induced inhibition of human plasma cholinesterase studied by the fluorescence spectroscopy method

NASA Astrophysics Data System (ADS)

Pavelkić, V. M.; Krinulović, K. S.; Savić, J. Z.; Ilić, M. A.

2008-05-01

The in vitro effect of technical grade malathion was assessed via the kinetic parameters of human plasma butyrylcholinesterase (BChE) using N-methylindoxyl acetate as a substrate for BChE. An inhibitor kinetics study demonstrated the existence of a biphasic inhibition curve, indicating high-and low-affinity binding sites of malathion. The IC 50 values as calculated from the experimental inhibition curves were 1.33 × 10-9 and 1.48 × 10-5 M for the high-and low-affinity binding sites, respectively; Hill’s analysis gave 1.29 × 10-9 and 1.38 × 10-6 M. The Cornish-Bowden plots and their secondary plots indicated that the nature of inhibition was of mixed type with the predominant competitive character of both affinity binding sites.

Spectroscopic studies on the binding interaction of phenothiazinium dyes, azure A and azure B to double stranded RNA polynucleotides

NASA Astrophysics Data System (ADS)

Khan, Asma Yasmeen; Suresh Kumar, Gopinatha

2016-01-01

This manuscript presents spectroscopic characterization of the interaction of two phenothiazinium dyes, azure A and azure B with double stranded (ds) ribonucleic acids, poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C). Absorbance and fluorescence studies revealed that these dyes bind to the RNAs with binding affinities of the order 106 M-1 to poly(A).poly(U), and 105 M-1 to poly(C).poly(G) and poly(I).poly(C), respectively. Fluorescence quenching and viscosity data gave conclusive evidence for the intercalation of the dyes to these RNA duplexes. Circular dichroism results suggested that the conformation of the RNAs was perturbed on interaction and the dyes acquired strong induced optical activity on binding. Azure B bound to all the three RNAs stronger than azure A and the binding affinity varied as poly(A).poly(U) > poly(C).poly(G) > poly(I).poly(C) for both dyes.

Sulfogalactosylglycerolipid is involved in human gamete interaction.

PubMed

Weerachatyanukul, W; Rattanachaiyanont, M; Carmona, E; Furimsky, A; Mai, A; Shoushtarian, A; Sirichotiyakul, S; Ballakier, H; Leader, A; Tanphaichitr, N

2001-12-01

Recent results from our laboratory have revealed the role of sulfogalactosylglycerolipid (SGG) in mouse sperm-zona pellucida (ZP) binding. In this report, we demonstrated the presence of SGG in Percoll-gradient centrifuged (PGC) human sperm by high performance thin layer chromatography with orcinol and Azure A staining, specific for glycolipids and sulfolipids, respectively. SGG in human PGC sperm was quantified by its affinity to Azure A to be 12-15 mol% of sperm lipids. Indirect immunofluorescence revealed that SGG existed on both live and aldehyde fixed human sperm in the head region. Pretreatment of human PGC sperm with affinity purified antiSGG Fab markedly inhibited sperm binding to the ZP in a concentration dependent manner, without any changes in the spontaneous acrosome rate or sperm motility parameters. Fluorescently labeled SGG liposomes also bound uniformly to isolated human ZP, while fluorescently labeled galactosylglycerolipid (GG, SGG's parental lipid) or phosphatidylserine (PS, negatively charged like SGG) liposomes did not. All of these results suggested the role of human sperm SGG in ZP binding. Copyright 2001 Wiley-Liss, Inc.

Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence.

PubMed

Zhu, Cuige; Zuo, Yinglin; Wang, Ruimin; Liang, Baoxia; Yue, Xin; Wen, Gesi; Shang, Nana; Huang, Lei; Chen, Yu; Du, Jun; Bu, Xianzhang

2014-08-14

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

NASA Astrophysics Data System (ADS)

Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

2015-03-01

Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems.

PubMed

Lattanzio, F A

1990-08-31

A novel method of determining the apparent dissociation constants of fluorescent calcium indicators is described which utilizes Chelex-100 ion exchange resin and 45Ca. The affinity for calcium of indicators fluo-3, fura-2 and indo-1 measured at either 22 degrees or 37 degrees C decreases as pH is decreased from 7.4 to 5.5. These measurements agree with determinations made using EDTA-calcium buffers. The 1:1 calcium:indicator complex is maintained under all conditions. The necessity to correct dissociation constants during intracellular acidification to properly interpret fluorescence measurements is illustrated by indo-1 measurements in the ischemic rat heart.

Investigation of the binding between pepsin and nucleoside analogs by spectroscopy and molecular simulation.

PubMed

Li, Zhen; Li, Zhigang; Yang, Lingling; Xie, Yuanzhe; Shi, Jie; Wang, Ruiyong; Chang, Junbiao

2015-03-01

In this paper, the interactions of pepsin with CYD (cytidine) or nucleoside analogs, including FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine) and CMP (cytidine monophosphate), were investigated by fluorescence, UV-visible absorption and synchronous fluorescence spectroscopy under mimic physiological conditions. The results indicated that FNC (CYD/CMP) caused the fluorescence quenching by the formation of complex. The binding constants and thermo-dynamic parameters at three different temperatures were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize the complex. The F atom in FNC might weaken the binding of nucleoside analog to pepsin. Results showed that CYD was the strongest quencher and bound to pepsin with higher affinity.

Influence of dyadic matching of affect on infant self-regulation.

PubMed

Noe, Daniela; Schluckwerder, Sabine; Reck, Corinna

2015-01-01

Affective behavioural matching during face-to-face interaction fosters the transition from mutual regulation to infant self-regulation. Optimum midrange models of mother-infant interaction hold that moderate degrees of dyadic matching facilitate infant socio-emotional development. The aim of this study was to examine which degree of dyadic matching is most beneficial for infant self-regulation. To evaluate this model, 3 groups of highly, midrange and poorly matched dyads were created from a mixed sample of 68 dyads with healthy and post-partum depressed mothers and their infants (age range = 1-8 months, mean age = 3.9 months). Mother-infant interactions were videotaped in the face-to-face still-face paradigm (FFSF) and micro-analytically coded. Specifically, the relation between affective behavioural matching in FFSF play and infant positive and negative affect in FFSF still face and FFSF reunion was explored. Contrary to our expectation, we found a monotonous trend for all groups: the more matching in FFSF play, the more positive and less negative affect the infant showed in FFSF still face and FFSF reunion, respectively. The present findings further illuminate the association between different degrees of dyadic matching in early mother-infant interaction and infant self-regulation. Further research should focus on the integration and replication of findings and conceptual approaches to further evaluate and refine the concept of midrange matching and make it applicable to therapeutic work with mothers and their infants.

FRET-based quantum dot immunoassay for rapid and sensitive detection of Aspergillus amstelodami.

PubMed

Kattke, Michele D; Gao, Elizabeth J; Sapsford, Kim E; Stephenson, Larry D; Kumar, Ashok

2011-01-01

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 10(3) spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 10(5) CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis.

Using a Fluorescent Cytosine Analogue tC[superscript o] To Probe the Effect of the Y567 to Ala Substitution on the Preinsertion Steps of dNMP Incorporation by RB69 DNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, Shuangluo; Beckman, Jeff; Wang, Jimin

2012-10-10

Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC{sup o}), to identify preinsertion step(s) altered by NBP flexibility. When tC{sup o} is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC{sup o} is also quenched in the presence of dATP. We determined the crystalmore » structure of the dATP/tC{sup o}-containing ternary complex of the RB69pol Y567A mutant at 1.9 {angstrom} resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC{sup o}. Stabilization of the dATP/tC{sup o} base pair involved movement of the tC{sup o} backbone sugar into the DNA minor groove and required tilting of the tC{sup o} tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity.« less

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

PubMed Central

Kattke, Michele D.; Gao, Elizabeth J.; Sapsford, Kim E.; Stephenson, Larry D.; Kumar, Ashok

2011-01-01

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 103 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 105 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis. PMID:22163961

Fluorometric determination of the activity of alkaline phosphatase based on the competitive binding of gold nanoparticles and pyrophosphate to CePO4:Tb nanorods.

PubMed

Xu, Ai-Zhen; Zhang, Li; Zeng, Hui-Hui; Liang, Ru-Ping; Qiu, Jian-Ding

2018-05-09

A fluorometric method is described for the determination of the activity of alkaline phosphatase (ALP). It relies on the competition between gold nanoparticles (AuNPs) and pyrophosphate (PPi) for the coordination sites on the surface of CePO 4 :Tb nanorods. The green fluorescence of the CePO 4 :Tb is reduced in the presence of AuNPs due to fluorescence resonance energy transfer (FRET), but can be restored on addition of PPi due to the stronger affinity of PPi to the CePO 4 :Tb. In the presence of ALP, PPi is hydrolyzed to form phosphate which has much weaker affinity for the CePO 4 :Tb. Hence, the AuNPs will reassemble on the CePO 4 :Tb, and fluorescence is reduced. Fluorescence drops linearly in the 0.2 to 100 U·L -1 activity range, and the detection limit is 60 mU·L -1 (at S/N = 3). The method does not require any modification of the surface of the CePO 4 :Tb and is highly sensitive and selective. The inhibition of ALP activity by Na 3 VO 4 was also studied. In our perception, the method may find application in the diagnosis of ALP-related diseases, in screening for inhibitors, and in studies on ALP-related functions in biological systems. Graphical abstract A assay for the detection of alkaline phosphatase is proposed based on the fluorescence resonance energy transfer between CePO 4 :Tb and AuNPs. It relies on the competitive binding of AuNPs and pyrophosphate (PPi) to CePO 4 :Tb and the hydrolysis of PPi by ALP.

Deciphering the complexation process of a fluoroquinolone antibiotic, levofloxacin, with bovine serum albumin in the presence of additives

NASA Astrophysics Data System (ADS)

Kaur, Amandeep; Khan, Imran Ahmd; Banipal, Parampaul Kaur; Banipal, Tarlok Singh

2018-02-01

The current work aims to explore the thermodynamic and conformational aspects for the binding of fluoroquinolone antibacterial drug, levofloxacin (LFC), with bovine serum albumin (BSA) using calorimetric, spectroscopic (UV-visible, fluorescence, circular dichroism, and 1H NMR), dynamic light scattering (DLS) and computational methods (molecular docking). The binding of LFC with BSA at two sequential sites with higher affinity ( 103 M- 1) at the first site has been explored by calorimetry whereas the binding at a single site with affinity of the order of 104 M- 1 has been observed from fluorescence spectroscopy. The calorimetric study in the presence of additives along with docking analysis reveals the significant role of electrostatic, hydrogen bonding, and hydrophobic interactions in the association process. The slight conformational changes in protein as well as the changes in the water network structure around the binding cavity of protein have been observed from spectroscopic and DLS measurements. The LFC induced quenching of BSA fluorescence was observed to be initiated mainly through the static quenching process and this suggests the formation of ground state LFC-BSA association complex. The stronger interactions of LFC in the cavity of Sudlow site I (subdomain IIA) of protein have been explored from site marker calorimetric and molecular docking study.

Boronic acid recognition of non-interacting carbohydrates for biomedical applications: increasing fluorescence signals of minimally interacting aldoses and sucralose†

PubMed Central

Resendez, Angel; Halim, Md Abdul; Singh, Jasmeet; Webb, Dominic-Luc

2017-01-01

To address carbohydrates that are commonly used in biomedical applications with low binding affinities for boronic acid based detection systems, two chemical modification methods were utilized to increase sensitivity. Modified carbohydrates were analyzed using a two component fluorescent probe based on boronic acid-appended viologen–HPTS (4,4′-o-BBV). Carbohydrates normally giving poor signals (fucose, l-rhamnose, xylose) were subjected to sodium borohydride (NaBH4) reduction in ambient conditions for 1 h yielding the corresponding sugar alcohols from fucose, l-rhamnose and xylose in essentially quantitative yields. Compared to original aldoses, apparent binding affinities were increased 4–25-fold. The chlorinated sweetener and colon permeability marker sucralose (Splenda), otherwise undetectable by boronic acids, was dechlorinated to a detectable derivative by reactive oxygen and hydroxide intermediates by the Fenton reaction or by H2O2 and UV light. This method is specific to sucralose as other common sugars, such as sucrose, do not contain any carbon-chlorine bonds. Significant fluorescence response was obtained for chemically modified sucralose with the 4,4′-o-BBV–HPTS probe system. This proof of principle can be applied to biomedical applications, such as gut permeability, malabsorption, etc. PMID:29130464

Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

NASA Astrophysics Data System (ADS)

Strzelczyk, Paweł; Bujacz, Grzegorz

2016-04-01

Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

Interaction of phenolic acids and their derivatives with human serum albumin: Structure-affinity relationships and effects on antioxidant activity.

PubMed

Zhang, Yunyue; Wu, Simin; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Ren, Fazheng; Zhang, Hao

2018-02-01

In this study, 111 phenolic acids and their derivatives were chosen to investigate their structure-affinity relationships when binding to human serum albumin (HSA), and effects on their antioxidant activity. A comprehensive mathematical model was employed to calculate the binding constants, using a fluorescence quenching method, and this was corrected for the inner-filter effect to improve accuracy. We found that a hydroxy group at the 2-position of the benzene ring exerted a positive effect on the affinities, while a 4-hydroxy substituent had a negative influence. Both methylation of the hydroxy groups and replacing the hydroxy groups with methyl groups at the 3- and 4-positions of the benzene ring enhanced the binding affinities. Hydrophobic force and hydrogen bonding were binding forces for the phenolic acids, and their methyl esters, respectively. The antioxidant activity of the HSA-phenolic acid interaction compounds was higher than that of the phenolic acids alone. Copyright © 2017. Published by Elsevier Ltd.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

PubMed Central

Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

2012-01-01

F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Surface-modified multifunctional MIP nanoparticles

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; Perez de Vargas Sansalvador, Isabel; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J.; Piletsky, Sergey

2013-04-01

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors. Electronic supplementary information (ESI) available: Details of the synthesis of eosin O-acrylate monomer and 1H-NMR spectrum of MIP NPs post-derivatised with PEG shell. See DOI: 10.1039/c3nr00354j

Anion sensing with a Lewis acidic BODIPY-antimony(v) derivative.

PubMed

Christianson, Anna M; Gabbaï, François P

2017-02-21

We describe the synthesis of a BODIPY dye substituted with a Lewis acidic antimony(v) moiety. This compound, which has been fully characterized, shows a high affinity for small anions including fluoride and cyanide, the complexation of which elicits a fluorescence turn-on response.

The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

PubMed

Hector, R F; Braun, P C; Hart, J T; Kamarck, M E

1990-01-01

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

PubMed

Alghamdi, Othman A; King, Nicola; Jones, Graham L; Moens, Pierre D J

2017-12-01

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C 3 -(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C 3 -(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule.

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane▿

PubMed Central

Kaserer, Wallace A.; Jiang, Xiaoxu; Xiao, Qiaobin; Scott, Daniel C.; Bauler, Matthew; Copeland, Daniel; Newton, Salete M. C.; Klebba, Phillip E.

2008-01-01

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB+ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins. PMID:18390658

G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging

NASA Astrophysics Data System (ADS)

Zhu, Jinbo; Zhang, Libing; Teng, Ye; Lou, Baohua; Jia, Xiaofang; Gu, Xiaoxiao; Wang, Erkang

2015-07-01

Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging. Electronic supplementary information (ESI) available: Experiment details, Tables S1-3 and Fig. S1-4. See DOI: 10.1039/c5nr03092g

Binding of RNA by the Nucleoproteins of Influenza Viruses A and B

PubMed Central

Labaronne, Alice; Swale, Christopher; Monod, Alexandre; Schoehn, Guy; Crépin, Thibaut; Ruigrok, Rob W. H.

2016-01-01

This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)6-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses. PMID:27649229

The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Digital Linear Tape (DLT) technology and product family overview

NASA Technical Reports Server (NTRS)

Lignos, Demetrios

1994-01-01

The demand that began a couple of years ago for increased data storage capacity continues. Peripheral Strategies (a Santa Barbara, California, Storage Market Research Firm) projects the amount of data stored on the average enterprise network will grow by 50 percent to 100 percent per year. Furthermore, Peripheral Strategies says that a typical mid-range workstation system containing 30GB to 50GB of storage today will grow at the rate of 50 percent per year. Dan Friedlander, a Boulder, Colorado-based consultant specializing in PC-LAN backup, says, 'The average NetWare LAN is about 8GB, but there are many that have 30GB to 300GB.....' The substantial growth of storage requirements has created various tape technologies that seek to satisfy the needs of today's and, especially, the next generations's systems and applications. There are five leading tape technologies in the market today: QIC (Quarter Inch Cartridge), IBM 3480/90, 8mm, DAT (Digital Audio Tape) and DLT (Digital Linear Tape). Product performance specifications and user needs have combined to classify these technologies into low-end, mid-range, and high-end systems applications. Although the manufacturers may try to position their products differently, product specifications and market requirements have determined that QIC and DAT are primarily low-end systems products while 8mm and DLT are competing for mid-range systems applications and the high-end systems space, where IBM compatibility is not required. The 3480/90 products seem to be used primarily in the IBM market, for interchangeability purposes. There are advantages and disadvantages for each of the tape technologies in the market today. We believe that DLT technology offers a significant number of very important features and specifications that make it extremely attractive for most current as well as emerging new applications, such as Hierarchical Storage Management (HSM). This paper will demonstrate why we think that the DLT technology and family of DLT products will become the technology of choice for most new applications in the mid-range and high-end (non-IBM) markets.

Thirty-fifth anniversary of the optical affinity sensor for glucose: a personal retrospective.

PubMed

Schultz, Jerome S

2015-01-01

Since 1962 when Clark introduced the enzyme electrode, research has been intense for a robust implantable glucose sensor. An alternative "optical affinity sensor" was introduced by Jerome Schultz in 1979. The evolution of this sensor technology into a new methodology is reviewed. The approach integrates a variety of disparate concepts: the selectivity of immunoassays-selectivity for glucose was obtained with concanavalin A, detection sensitivity was obtained with fluorescence (FITC-Dextran), and miniaturization was achieved by the use of an optical fiber readout system. Refinements of Schultz's optical affinity sensor approach over the past 35 years have led to a number of configurations that show great promise to meet the needs of a successful implantable continuous monitoring device for diabetics, some of which are currently being tested clinically. © 2014 Diabetes Technology Society.

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity.

PubMed

Cheng, Y; Lin, H; Xue, D; Li, R; Wang, K

2001-02-14

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.

The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.

PubMed

Imanaka, Hiroyuki; Yamadzumi, Daisuke; Yanagita, Keisuke; Ishida, Naoyuki; Nakanishi, Kazuhiro; Imamura, Koreyoshi

2016-03-01

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016. © 2016 American Institute of Chemical Engineers.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.

PubMed

Kirchberg, Kristina; Kim, Tai-Yang; Möller, Martina; Skegro, Darko; Dasara Raju, Gayathri; Granzin, Joachim; Büldt, Georg; Schlesinger, Ramona; Alexiev, Ulrike

2011-11-15

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

PubMed Central

Kirchberg, Kristina; Kim, Tai-Yang; Möller, Martina; Skegro, Darko; Dasara Raju, Gayathri; Granzin, Joachim; Büldt, Georg; Schlesinger, Ramona; Alexiev, Ulrike

2011-01-01

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the “phosphate-sensor” of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins. PMID:22039220

Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

NASA Astrophysics Data System (ADS)

Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

2017-10-01

Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

PubMed

Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

2016-01-01

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

PubMed

Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

2015-10-07

Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2015-02-24

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and similar to 90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context.« less

[The management of the metastatic disease by the surgeons].

PubMed

Mery, Eliane; Golzio, Muriel; Thibault, Benoît; Ferron, Gwenael; Querleu, Denis; Delord, Jean-Pierre; Couderc, Bettina

2013-04-01

The high rate of recurrence after apparently complete surgery and/or complete clinical response to chemotherapy implies that most patients have undetected minimal residual disease. Novel techniques such as laparoscopic or laparotomic fluorescence may prove to be crucial in reassessing the definition of primary outcome in ovarian cancer management. For this purpose, the importance of fluorescence in the peroperative detection of human ovarian adenocarcinoma cells has to be validated in animal models prior to be used in clinic by determining its efficiency in detecting infra-millimetric tumor metastases. In the preclinical data presented, a fluorescent RAFT-(cRGD)4 tracer molecule (AngioStamp(®)) with a very high affinity for the αvβ3 integrin was used. Infrared fluorescence was visualized with Fluobeam(®), an open fluorescent imaging system that could potentially be used in peroperative conditions in the future. We showed that this novel technique allowed the specific detection of residual tumor deposits and inframillimetric metastases in mice and dogs.

Novel fluorogenic probe for fluoride ion based on the fluoride-induced cleavage of tert-butyldimethylsilyl ether

NASA Astrophysics Data System (ADS)

Yang, Xiao-Feng

2007-06-01

A highly sensitive and selective fluorogenic probe for fluoride ion, 4-methylumbelliferyl tert-butyldimethylsilyl ether (4-MUTBS), was designed and synthesized. 4-MUTBS was a weakly fluorescent compound and was synthesized via the one-step reaction of 4-MU with tert-butyldimethylsilyl chloride. Upon incubation with fluoride ion in acetone-water solution (7:3, v/v), the Si-O bond of 4-MUTBS was cleaved and highly fluorescent 4-methylumbelliferone (4-MU) was released, hence leading to the fluorescence increase of the reaction solution. The fluorescence increase is linearly with fluoride concentration in the range 50-8000 nmol l -1 with a detection limit of 19 nmol l -1 (3 σ). Because of the high affinity of silicon toward fluoride ion, the proposed probe shows excellent selectivity toward fluoride ion over other anions. The method has been successfully applied to the fluoride determination in toothpaste and tap water samples.

Highly selectively monitoring heavy and transition metal ions by a fluorescent sensor based on dipeptide.

PubMed

Neupane, Lok Nath; Thirupathi, Ponnaboina; Jang, Sujung; Jang, Min Jung; Kim, Jung Hwa; Lee, Keun-Hyeung

2011-09-15

Fluorescent sensor (DMH) based on dipeptide was efficiently synthesized in solid phase synthesis. The dipeptide sensor shows sensitive response to Ag(I), Hg(II), and Cu(II) among 14 metal ions in 100% aqueous solution. The fluorescent sensor differentiates three heavy metal ions by response type; turn on response to Ag(I), ratiometric response to Hg(II), and turn off detection of Cu(II). The detection limits of the sensor for Ag(I) and Cu(II) were much lower than the EPA's drinking water maximum contaminant levels (MCL). Specially, DMH penetrated live cells and detected intracellular Ag(+) by turn on response. We described the fluorescent change, binding affinity, detection limit for the metal ions. The study of a heavy metal-responsive sensor based on dipeptide demonstrates its potential utility in the environment field. Copyright © 2011 Elsevier B.V. All rights reserved.

Interaction of fluorescent sensor with superparamagnetic iron oxide nanoparticles.

PubMed

Karunakaran, Chockalingam; Jayabharathi, Jayaraman; Sathishkumar, Ramalingam; Jayamoorthy, Karunamoorthy

2013-06-01

To sense superparamagnetic iron oxides (Fe2O3 and Fe3O4) nanocrystals a sensitive bioactive phenanthroimidazole based fluorescent molecule, 2-(4-fluorophenyl)-1-phenyl-1H-phenanthro [9,10-d] imidazole has been designed and synthesized. Electronic spectral studies show that phenanthroimidazole is bound to the surface of iron oxide semiconductors. Fluorescent enhancement has been explained on the basis of photo-induced electron transfer (PET) mechanism and apparent binding constants have been deduced. Binding of phenanthroimidazole with iron oxide nanoparticles lowers the HOMO and LUMO energy levels of phenanthroimidazole molecule. Chemical affinity between the nitrogen atom of the phenanthroimidazole and Fe(2+) and Fe(3+) ions on the surface of the nano-oxide may result in strong binding of the phenanthroimidazole derivative with the nanoparticles. The electron injection from the photoexcited phenanthroimidazole to the iron oxides conduction band explains the enhanced fluorescence. Copyright © 2013 Elsevier B.V. All rights reserved.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

PubMed

Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

PubMed Central

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining wasmore » determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.« less

Novel Benzothiazole Derivatives as Fluorescent Probes for Detection of β-Amyloid and α-Synuclein Aggregates.

PubMed

Watanabe, Hiroyuki; Ono, Masahiro; Ariyoshi, Taisuke; Katayanagi, Rikako; Saji, Hideo

2017-08-16

Deposits of β-amyloid (Aβ) and α-synuclein (α-syn) are the hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. The detection of these protein aggregates with fluorescent probes is particularly of interest for preclinical studies using fluorescence microscopy on human brain tissue. In this study, we newly designed and synthesized three push-pull benzothiazole (PP-BTA) derivatives as fluorescent probes for detection of Aβ and α-syn aggregates. Fluorescence intensity of all PP-BTA derivatives significantly increased upon binding to Aβ(1-42) and α-syn aggregates in solution. In in vitro saturation binding assays, PP-BTA derivatives demonstrated affinity for both Aβ(1-42) (K d = 40-148 nM) and α-syn (K d = 48-353 nM) aggregates. In particular, PP-BTA-4 clearly stained senile plaques composed of Aβ aggregates in the AD brain section. Moreover, it also labeled Lewy bodies composed of α-syn aggregates in the PD brain section. These results suggest that PP-BTA-4 may serve as a promising fluorescent probe for the detection of Aβ and α-syn aggregates.

Synthesis of fluorescent dye-doped silica nanoparticles for target-cell-specific delivery and intracellular microRNA imaging.

PubMed

Li, Henan; Mu, Yawen; Qian, Shanshan; Lu, Jusheng; Wan, Yakun; Fu, Guodong; Liu, Songqin

2015-01-21

MicroRNA (miRNA) is found to be up-regulated in many kinds of cancer and therefore is classified as an oncomiR. Herein, we design a multifunctional fluorescent nanoprobe (FSiNP-AS/MB) with the AS1411 aptamer and a molecular beacon (MB) co-immobilized on the surface of the fluorescent dye-doped silica nanoparticles (FSiNPs) for target-cell-specific delivery and intracellular miRNA imaging. The FSiNPs were prepared by a facile reverse microemulsion method from tetraethoxysilane and silane derivatized coumarin that was previously synthesized by click chemistry. The as-prepared FSiNPs possess uniform size distribution, good optical stability and biocompatibility. In addition, there is a remarkable affinity interaction between the AS1411 aptamer and the nucleolin protein on the cancer cell surface. Thus, a target-cell-specific delivery system by the FSiNP-AS/MB is proposed for effectively transferring a MB into the cancer cells to recognize the target miRNA. Using miRNA-21 in MCF-7 cells (a human breast cancer cell line) as a model, the proposed multifunctional nanosystems not only allow target-cell-specific delivery with the binding affinity of AS1411, but also can track simultaneously the transfected cells and detect intracellular miRNA in situ. The proposed multifunctional nanosystems are a promising platform for a highly sensitive luminescent nonviral vector in biomedical and clinical research.

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

PubMed Central

Szmacinski, Henryk; Toshchakov, Vladimir; Lakowicz, Joseph R.

2014-01-01

Abstract. Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population. PMID:24770662

The effect of Berberine on the secondary structure of human serum albumin

NASA Astrophysics Data System (ADS)

Li, Ying; He, WenYing; Tian, Jianniao; Tang, Jianghong; Hu, Zhide; Chen, Xingguo

2005-05-01

The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is designed to examine the effect of Berberine (an ancient Chinese drug used for antimicrobial, antiplasmodial, antidiarrheal and cardiovascular) on the solution structure of HSA using fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopic methods. The fluorescence spectroscopic results show that the fluorescence intensity of HSA was significantly decreased in the presence of Berberine. The Scatchard's plots indicated that the binding of Berberine to HSA at 296, 303, 318 K is characterized by one binding site with the binding constant is 4.071(±0.128)×10 4, 3.741(±0.089)×10 4, 3.454(±0.110)×10 4 M -1, respectively. The protein conformation is altered (FT-IR and CD data) with reductions of α-helices from 54 to 47% for free HSA to 45-32% and with increases of turn structure5% for free HSA to 18% in the presence of Berberine. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, Berberine bound to HSA was mainly based on hydrophobic interaction and electrostatic interaction cannot be excluded from the binding. Furthermore, the displace experiments indicate that Berberine can bind to the subdomain IIA, that is, high affinity site (site II).

Amyloid-β Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

PubMed

Fu, Hualong; Tu, Peiyu; Zhao, Liu; Dai, Jiapei; Liu, Boli; Cui, Mengchao

2016-02-02

The formation of extracellular amyloid-β (Aβ) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aβ plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target Aβ plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aβ aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aβ plaques in the brain.

Cytidine-stabilized gold nanocluster as a fluorescence turn-on and turn-off probe for dual functional detection of Ag(+) and Hg(2+).

PubMed

Zhang, Yuanyuan; Jiang, Hui; Wang, Xuemei

2015-04-22

In this study, we have developed a label-free, dual functional detection strategy for highly selective and sensitive determination of aqueous Ag(+) and Hg(2+) by using cytidine stabilized Au NCs and AuAg NCs as fluorescent turn-on and turn off probes, respectively. The Au NCs and AuAg NCs showed a remarkably rapid response and high selectivity for Ag(+) and Hg(2+) over other metal ions, and relevant detection limit of Ag(+) and Hg(2+) is ca. 10 nM and 30 nM, respectively. Importantly, the fluorescence enhanced Au NCs by doping Ag(+) can be conveniently reusable for the detection of Hg(2+) based on the corresponding fluorescence quenching. The sensing mechanism was based on the high-affinity metallophilic Hg(2+)-Ag(+) interaction, which effectively quenched the fluorescence of AuAg NCs. Furthermore, these fluorescent nanoprobes could be readily applied to Ag(+) and Hg(2+) detection in environmental water samples, indicating their possibility to be utilized as a convenient, dual functional, rapid response, and label-free fluorescence sensor for related environmental and health monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of the bioactive component Aloe dihydroisocoumarin with human serum albumin

NASA Astrophysics Data System (ADS)

Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Tang, Ya-Lin

2008-11-01

Aloe dihydroisocoumarin, one of new components isolated from Aloe vera, can scavenge reactive oxygen species. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydroisocoumarin with human serum albumin (HSA) has been investigated by using fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydroisocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. An isoemissive point at 414 nm is seen, indicating that the quenching of HSA fluorescence depends on the formation of Aloe dihydroisocoumarin-HSA complex, which is further confirmed by fluorescence dynamic result. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydroisocoumarin with HSA causes a conformational change of the protein, with the gain of α-helix, β-sheet and random coil stability and the loss of β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FTIR experiments along with the docking studies suggest that Aloe dihydroisocoumarin binds to residues located in subdomain IIA of HSA.

Evaluation of four affibody-based near-infrared fluorescent probes for optical imaging of epidermal growth factor receptor positive tumors.

PubMed

Qi, Shibo; Miao, Zheng; Liu, Hongguang; Xu, Yingding; Feng, Yaqing; Cheng, Zhen

2012-06-20

The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-ZEGFR:1907 and Alex680-ZEGFR:1907 displayed higher tumor-to-normal tissue ratios than those of the other two probes. This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-ZEGFR:1907 and Cy5.5-ZEGFR:1907 are excellent candidates as EGFR-targeted probes for optical imaging.

Probing the binding of flavonoids to catalase by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Zhu, Jingfeng; Zhang, Xia; Li, Daojin; Jin, Jing

2007-10-01

The binding of flavonoids (quercetin and myricetin) to catalase was investigated by fluorescence and circular dichroism (CD) techniques under physiological conditions. The binding parameters and binding mode between flavonoids and catalase were determined, and the results of synchronous fluorescence spectra and CD indicated a conformational change of catalase with addition of flavonoids. The effect of both Cu 2+ and vitamin C on the binding constant of flavonoid-catalase was also examined. The experiment data show that the difference of the structure characteristics of quercetin and myricetin has a significant effect on their binding affinity for catalase.

Description of the pupa of the Himalayan mosquito, Ochlerotatus pulchriventer.

PubMed

Darsie, Richard F

2009-09-01

The pupa of Ochlerotatus pulchriventer (=Aedes pulchriventer) is described and illustrated for the first time. It was collected at midrange altitudes in the Himalaya Mountains of Nepal. A subspecies from Taiwan is discussed.

Binding modes of environmental endocrine disruptors to human serum albumin: insights from STD-NMR, ITC, spectroscopic and molecular docking studies.

PubMed

Yang, Hongqin; Huang, Yanmei; Liu, Jiuyang; Tang, Peixiao; Sun, Qiaomei; Xiong, Xinnuo; Tang, Bin; He, Jiawei; Li, Hui

2017-09-11

Given that bisphenols have an endocrine-disrupting effect on human bodies, thoroughly exposing their potential effects at the molecular level is important. Saturation transfer difference (STD) NMR-based binding studies were performed to investigate the binding potential of two bisphenol representatives, namely, bisphenol B (BPB) and bisphenol E (BPE), toward human serum albumin (HSA). The relative STD (%) suggested that BPB and BPE show similar binding modes and orientations, in which the phenolic rings were spatially close to HSA binding site. ITC analysis results showed that BPB and BPE were bound to HSA with moderately strong binding affinity through electrostatic interactions and hydrogen bonds. The order of binding affinity of HSA for two test bisphenols is as follows: BPE > BPB. The results of fluorescence competitive experiments using 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as competitors, combined with molecular docking indicated that both bisphenols are prone to attach to the binding site II in HSA. Spectroscopic results (FT-IR, CD, synchronous and 3D fluorescence spectra) showed that BPB/BPE induces different degrees of microenvironmental and conformational changes to HSA.

Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains.

PubMed

Anumala, Upendra Rao; Gu, Jiamin; Lo Monte, Fabio; Kramer, Thomas; Heyny-von Haußen, Roland; Hölzer, Jana; Goetschy-Meyer, Valerie; Schön, Christian; Mall, Gerhard; Hilger, Ingrid; Czech, Christian; Herms, Jochen; Schmidt, Boris

2013-09-01

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization. Copyright © 2013 Elsevier Ltd. All rights reserved.

A novel method for the study of molecular interaction by using microscale thermophoresis.

PubMed

Mao, Yexuan; Yu, Lanlan; Yang, Ran; Qu, Ling-bo; Harrington, Perter de B

2015-01-01

The fundamental studies for the binding events of protein and its partner are crucial in drug development. In this study, a novel technology named microscale thermophoresis (MST) was applied in the investigation of molecular interaction between an organic dye fluorescein isothiocyanate (FITC) and bovine serum albumin (BSA), and the results were compared with those obtained from conventional fluorescence spectroscopy. The MST data demonstrated that with a short interaction time, FITC showed a high binding affinity for BSA by weak interaction instead of labeling the protein. By using competitive strategies in which warfarin and ibuprofen acted as the site markers of BSA, FITC was proven to mainly bind to the hydrophobic pocket of site II of BSA compared to site I of BSA. Except for the binding affinity, MST also provided additional information with respect to the aggregation of BSA and the binding of FITC to BSA aggregates, which is unobtainable by fluorescence spectroscopy. This work proves that MST as a new approach is powerful and reliable for investigation of protein-small molecule interaction. Copyright © 2014 Elsevier B.V. All rights reserved.

Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

PubMed

Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

2016-01-12

We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Facile Synthesis of Molecularly Imprinted Graphene Quantum Dots for the Determination of Dopamine with Affinity-Adjustable.

PubMed

Zhou, Xi; Wang, Anqi; Yu, Chenfei; Wu, Shishan; Shen, Jian

2015-06-10

A facilely prepared fluorescence sensor was developed for dopamine (DA) determination based on polyindole/graphene quantum dots molecularly imprinted polymers (PIn/GQDs@MIPs). The proposed sensor exhibits a high sensitivity with a linear range of 5 × 10(-10) to 1.2 × 10(-6) M and the limit of detection as low as 1 × 10(-10) M in the determination of DA, which is probably due to the tailor-made imprinted cavities for binding DA thought hydrogen bonds between amine groups of DA and oxygen-containing groups of the novel composite. Furthermore, the prepared sensor can rebind DA in dual-type: a low affinity type (noncovalent interaction is off) and a high affinity type (noncovalent interaction is on), and the rebinding interaction can be adjusted by tuning the pH, which shows a unique potential for adjusting the binding interaction while keeping the specificity, allowing for wider applications.

Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krejciríková, Veronika; Pachl, Petr; Fábry, Milan

2011-11-18

Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{submore » d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.« less

Rabbit anti-rabies immunoglobulins production and evaluation.

PubMed

Liu, Xinjian; Liu, Qiongqiong; Feng, Xiaomin; Tang, Qi; Wang, Zhongcan; Li, Suqing; Feng, Zhenqing; Zhu, Jin; Guan, Xiaohong

2011-04-01

Due to the disadvantages of human and equine rabies immunoglobulin, it is necessary to develop a substitute for HRIG and ERIG, especially for those people living in the developing countries. Because of higher affinity and lower immunogenicity of rabbit's immunoglobulins, anti-rabies immunoglobulins specific to rabies virus were produced in rabbits as a bioreactor, and had been characterized by ELISA, affinity assay, immunofluorescence assay (IFA), immunocytochemistry, rapid fluorescent focus inhibition test (RFFIT). ELISA, affinity assay and IFA showed that rabbit RIG (RRIG) bound specifically to rabies virions. RFFIT result showed that RRIG has neutralization activity. This result was confirmed in vivo in a Kunming mouse challenge model and the protection rate of the treatment with RRIG was higher (25%) than that offered by HRIG when mice were challenged with a lethal RV dose. Our results demonstrate that RRIG is safe and efficacious as a candidate drug to replace rabies immunoglobulin in post-exposure prophylaxis.

Surface-modified multifunctional MIP nanoparticles.

PubMed

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; Perez de Vargas Sansalvador, Isabel; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J; Piletsky, Sergey

2013-05-07

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.

Structure-affinity relationship of the interaction between phenolic acids and their derivatives and β-lactoglobulin and effect on antioxidant activity.

PubMed

Wu, Simin; Zhang, Yunyue; Ren, Fazheng; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Zhang, Hao

2018-04-15

In this study, 71 phenolic acids and their derivatives were used to investigate the structure-affinity relationship of β-lactoglobulin binding, and the effect of this interaction on antioxidant activity. Based on a fluorescence quenching method, an improved mathematical model was adopted to calculate the binding constants, with a correction for the inner-filter effect. Hydroxylation at the 3-position increased the affinity of the phenolic acids for β-lactoglobulin, while hydroxylation at the 2- or 4-positions had a negative effect. Complete methylation of all hydroxy groups, except at the 3-position, enhanced the binding affinity. Replacing the hydroxy groups with methyl groups at the 2-position also had a positive effect. Hydrogen bonding was one of the binding forces for the interaction. The antioxidant activity of phenolic acid-β-lactoglobulin complexes was higher than that of phenolic acids alone. These findings provide an understanding of the structure-activity relationship of the interaction between β-lactoglobulin and phenolic acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of Effective Fragment Potential Methos to the Redox Potential of Green Fluorescent Protein

NASA Astrophysics Data System (ADS)

Ghosh, Debashree; Krylov, Anna I.

2011-06-01

Green fluorescent proteins (GFP) can be considered as a model for flurogenic dyes and are of importance in photovoltaic materials. It exhibits bright green fluorescence when exposed to blue light and has been an extremely powerful tool as non-invasive marker in living cells and extensibly used in molecular and cell biology. The understanding of the underlying electronic structure of these proteins and its chromophore is therefore crucial to the understanding of the mechanism for its optical properties. The chromophore of the GFP is p-hydroxybenzylidene-imidazolinone (HBDI) and is embedded in the center of the β barrel of the GFP. Calculating redox potential of this chromophore is a challenging problem, especially in diverse solvents and protein environment. It is possible to carry out high-level accurate ab-initio calculation of ionization potential or electron affinity of the microsolvated chromophore or the bare chromophore. But, it is not possible to extend these calculations to bulk solvents due to the high computational cost. Effective fragment potential (EFP)[1,2] method gives us a convenient tool to understand such systems. In our work, we have benchmarked the ionization energy and electron affinity of the microsolvated GFP chromophore calculated by combined EOM-IP-CCSD/EFP and EOM-EA-CCSD/EFP with the EOM-IP-CCSD and EOM-EA-CCSD calculations of the oxidized and reduced forms. We have carried out similar EFP-EOM-IP-CCSD and EFP-EOM-EA-CCSD calculations of ionization potential and electron affinity of GFP choromophore in bulk solvent generated by ab-initio molecular dynamics simulations. [1] M. S. Gordon, L. Slipchenko, H. Li, J. H. Jensen, Annual Reports in Computational Chemistry, Volume 3, 177 (2007). [2] D. Ghosh, D. Kosenkov, V. Vanovschi, C.F. Williams, J.M. Herbert, M.S. Gordon, M.W. Schmidt, L.V. Slipchenko, and A.I. Krylov, J. Phys. Chem. A 114, 12739 (2010).

Determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods by affinity column cleanup and LC fluorescence detection.

PubMed

Senyuva, Hamide Z; Cimen, Dilek; Gilbert, John

2009-01-01

The effectiveness of an affinity column cleanup procedure followed by LC with fluorescence detection was established for the determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods. Traditional foods, such as baklava (finely layered pastry filled with nuts and steeped in syrup), halvah (containing sesame paste and pistachios), cevizli sucuk (a confection made of grape juice boiled and dried on strings of nuts), Turkish delight (containing hazelnuts, pistachios, or walnuts), and pişmaniye (candy made of sugar, butter, and flour), were tested, and the performance of the method was established with spiked samples. To examine the robustness of the methodology, baklava was prepared from raw materials and spiked at the initial stage of dry ingredients and through subsequent stages of preparation of dough, after cooking, and after addition of syrup and nuts. For all products, the analytical method required grinding the composite foodstuff under liquid nitrogen to form a fine powder, which was then thoroughly mixed before subsampling. After vortex extraction into methanol-water (aflatoxins) and aqueous sodium bicarbonate (ochratoxin A), the sample was filtered, diluted with phosphate-buffered saline, and then passed through either an aflatoxin or ochratoxin A affinity column before HPLC analysis with fluorescence detection (using post-column bromination for the aflatoxins). In all the traditional Turkish products, the recovery of aflatoxin B1 ranged from 77 to 98%, and LODs were <0.1 microg/kg. For ochratoxin A, the recoveries were from 88 to 93% and LODs were similarly <0.1 microLg/kg. Despite the complex nature of these traditional Turkish foods, which frequently contain products from sugar caramelization, there was no evidence of any interfering co-extractives, and the method has proved to be robust enough to be used for food control purposes.

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki

Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer inmore » living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.« less

Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.

PubMed

Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

2012-08-24

Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.

Heart Failure with Recovered EF and Heart Failure with Mid-Range EF: Current Recommendations and Controversies.

PubMed

Unkovic, Peter; Basuray, Anupam

2018-04-03

This review explores key features and potential management controversies in two emerging populations in heart failure: heart failure with recovered ejection fraction (HF-recovered EF) and heart failure with mid-range ejection fraction (HFmrEF). While HF-recovered EF patients have better outcomes than heart failure with reduced ejection fraction (HFrEF), they continue to have symptoms, persistent biomarker elevations, and abnormal outcomes suggesting a continued disease process. HFmrEF patients appear to have features of HFrEF and heart failure with preserved ejection fraction (HFpEF), but have a high prevalence of ischemic heart disease and may represent a transitory phase between the HFrEF and HFpEF. Management strategies have insufficient data to warrant standardization at this time. HF-recovered EF and HFmrEF represent new populations with unmet needs and expose the pitfalls of an EF basis for heart failure classification.

Near infrared fluorescent trypsin stabilized gold nanoclusters as surface plasmon enhanced energy transfer biosensor and in vivo cancer imaging bioprobe.

PubMed

Liu, Jing-Min; Chen, Jia-Tong; Yan, Xiu-Ping

2013-03-19

The simplicity of the green-synthesized routine and the availability of surface modification of diverse bioactive molecules make noble metal nanostructures highly suitable as multifunctional biomaterials for biological and biomedical application. Here, we report the preparation of trypsin stabilized gold nanoclusters (try-AuNCs) with near-infrared fluorescence for biosensing heparin based on surface plasmon enhanced energy transfer (SPEET) and folic acid (FA) modified try-AuNCs for in vivo cancer bioimaging. The SPEET/try-AuNCs fluorescence biosensor was designed via heparin mediated energy transfer between try-AuNCs and cysteamine modified gold nanoparticles (cyst-AuNPs). The developed SPEET/try-AuNCs fluorescence biosensor allowed sensitive and selective detection of heparin with a linear range of 0.1-4.0 μg mL(-1) and a detection limit (3s) of 0.05 μg mL(-1). The relative standard deviation for eleven replicate detections of 2.5 μg mL(-1) heparin was 1.1%, and the recoveries of the spiked heparin in human serum samples ranged from 97% to 100%. In addition, folic acid was immobilized on the surface of try-AuNCs to ameliorate the specific affinity of AuNCs for tumors, and the near-infrared fluorescent FA-try-AuNCs were applied for in vivo cancer imaging of high folate receptor (FR) expressing Hela tumor. In vivo study of the dynamic behavior and targeting ability of FA-try-AuNCs probe to Hela tumor bearing mice and normal nude mice validated the high specific affinity of FA-try-AuNCs probe to FR positive tumors. The results show that the prepared try-AuNCs have great potential as multifunctional biomaterials for biosensing biomolecules with SPEET mode and in vivo cancer imaging with high targeting ability.

Smart near-infrared fluorescence probes with donor-acceptor structure for in vivo detection of β-amyloid deposits.

PubMed

Cui, Mengchao; Ono, Masahiro; Watanabe, Hiroyuki; Kimura, Hiroyuki; Liu, Boli; Saji, Hideo

2014-03-05

The deposition of β-amyloid (Aβ) plaques in the parenchymal and cortical brain is accepted as the main pathological hallmark of Alzheimer's disease (AD); however, early detection of AD still presents a challenge. With the assistance of molecular imaging techniques, imaging agents specifically targeting Aβ plaques in the brain may lead to the early diagnosis of AD. Herein, we report the design, synthesis, and evaluation of a series of smart near-infrared fluorescence (NIRF) imaging probes with donor-acceptor architecture bridged by a conjugated π-electron chain for Aβ plaques. The chemical structure of these NIRF probes is completely different from Congo Red and Thioflavin-T. Probes with a longer conjugated π system (carbon-carbon double bond) displayed maximum emission in PBS (>650 nm), which falls in the best range for NIRF probes. These probes were proved to have affinity to Aβ plaques in fluorescent staining of brain sections from an AD patient and double transgenic mice, as well as in an in vitro binding assay using Aβ(1-42) aggregates. One probe with high affinity (K(i) = 37 nM, K(d) = 27 nM) was selected for in vivo imaging. It can penetrate the blood-brain barrier of nude mice efficiently and is quickly washed out of the normal brain. Moreover, after intravenous injection of this probe, 22-month-old APPswe/PSEN1 mice exhibited a higher relative signal than control mice over the same period of time, and ex vivo fluorescent observations confirmed the existence of Aβ plaques. In summary, this probe meets most of the requirements for a NIRF contrast agent for the detection of Aβ plaques both in vitro and in vivo.

Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

PubMed

Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

2007-05-01

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

In vivo protein stabilization based on fragment complementation and a split GFP system.

PubMed

Lindman, Stina; Hernandez-Garcia, Armando; Szczepankiewicz, Olga; Frohm, Birgitta; Linse, Sara

2010-11-16

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

PubMed

Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B

2008-10-01

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

PubMed

Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

2017-06-23

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

Landscape characteristics of Rhizophora mangle forests and propagule deposition in coastal environments of Florida (USA)

USGS Publications Warehouse

Sengupta, R.; Middleton, B.; Yan, C.; Zuro, M.; Hartman, H.

2005-01-01

Field dispersal studies are seldom conducted at regional scales even though reliable information on mid-range dispersal distance is essential for models of colonization. The purpose of this study was to examine the potential distance of dispersal of Rhizophora mangle propagules by comparing deposition density with landscape characteristics of mangrove forests. Propagule density was estimated at various distances to mangrove sources (R. mangle) on beaches in southwestern Florida in both high-and low-energy environments, either facing open gulf waters vs. sheltered, respectively. Remote sensing and Geographic Information Systems were used to identify source forests and to determine their landscape characteristics (forest size and distance to deposition area) for the regression analyses. Our results indicated that increasing density of propagules stranded on beaches was related negatively to the distance of the deposition sites from the nearest stands of R. mangle and that deposition was greatly diminished 2 km or more from the source. Measures of fragmentation such as the area of the R. mangle forests were related to propagule deposition but only in low-energy environments. Our results suggest that geographic models involving the colonization of coastal mangrove systems should include dispersal dynamics at mid-range scales, i.e., for our purposes here, beyond the local scale of the forest and up to 5 km distant. Studies of mangrove propagule deposition at various spatial scales are key to understanding regeneration limitations in natural gaps and restoration areas. Therefore, our study of mid-range propagule dispersal has broad application to plant ecology, restoration, and modeling. ?? Springer 2005.

Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

A fully-integrated aptamer-based affinity assay platform for monitoring astronaut health in space.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xianbin; Durland, Ross H.; Hecht, Ariel H.

2010-07-01

Here we demonstrate the suitability of robust nucleic acid affinity reagents in an integrated point-of-care diagnostic platform for monitoring proteomic biomarkers indicative of astronaut health in spaceflight applications. A model thioaptamer targeting nuclear factor-kappa B (NF-{kappa}B) is evaluated in an on-chip electrophoretic gel-shift assay for human serum. Key steps of (i) mixing sample with the aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated upstream of fluorescence-based detection. Challenges due to (i) nonspecific interactions with serum, and (ii) preconcentration at a nanoporous membrane are discussed and successfully resolved to yield a robust, rapid, and fully-integrated diagnostic system.

Organization of fluorescent cholesterol analogs in lipid bilayers - lessons from cyclodextrin extraction.

PubMed

Milles, Sigrid; Meyer, Thomas; Scheidt, Holger A; Schwarzer, Roland; Thomas, Lars; Marek, Magdalena; Szente, Lajos; Bittman, Robert; Herrmann, Andreas; Günther Pomorski, Thomas; Huster, Daniel; Müller, Peter

2013-08-01

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor. Copyright © 2013 Elsevier B.V. All rights reserved.

Engineering an FMN-based iLOV protein for the detection of arsenic ions.

PubMed

Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Lee, Chong-Soon; Yun, Hyungdon

2017-05-15

Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As 3+ ions. iLOV N468S exhibited an improved binding affinity with a dissociation constant of 1.5 μM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach. Copyright © 2017 Elsevier Inc. All rights reserved.

A fluorescent aptasensor based on a DNA pyramid nanostructure for ultrasensitive detection of ochratoxin A.

PubMed

Nameghi, Morteza Alinezhad; Danesh, Noor Mohammad; Ramezani, Mohammad; Hassani, Faezeh Vahdati; Abnous, Khalil; Taghdisi, Seyed Mohammad

2016-08-01

Analytical techniques for detection of ochratoxin A (OTA) in food products and blood serum are of great significance. In this study, a fluorescent aptasensor was developed for sensitive and specific detection of OTA, based on a DNA pyramid nanostructure (DPN) and PicoGreen (PG) dye. The designed aptasensor inherits characteristics of DPN, such as high stability and capacity for PG loading. PG, as a fluorescent dye, could bind to double-stranded DNA (dsDNA). In the absence of OTA, the pyramid structure of DPN remains intact, leading to a very strong fluorescence emission. Because of higher affinity of aptamer for its target relative to its complementary strand, upon addition of target, the pyramid structure of DPN is disassembled, leading to a weak fluorescence emission. The presented aptasensor showed high specificity toward OTA with a limit of detection (LOD) as low as 0.135 nM. Besides, the designed sensing strategy was successfully utilized to recognize OTA in serum and grape juice with LODs of 0.184 and 0.149 nM, respectively.

Characterization and validation of fluorescent receptor ligands: a case study of the ionotropic serotonin receptor.

PubMed

Hovius, Ruud

2013-01-01

The application of fluorescent receptor ligands has become widespread, incited by two important reasons. "Seeing is believing"-it is possible to visualize in real time in live cells ligand-receptor interactions, and to locate the receptors with subcellular precision allowing one to follow, e.g., internalization of the ligand-receptor complex. The high sensitivity of photon detection permits observation of on the one hand receptor-ligand interactions on cells with low, native receptor abundance, and on the other of individual fluorophores unveiling the stochastic properties of single ligand-receptor complexes.The major bottlenecks that impede extensive use of fluorescent ligands are due to possible dramatic changes of the pharmacological properties of a ligand upon chemical modification and fluorophore conjugation, aggravated by the observation that different fluorophores can provoke very dissimilar effects. This makes it virtually impossible to predict beforehand which labelling strategy to use to produce a fluorescent ligand with the desired qualities.Here, we focus on the design, synthesis, and evaluation of a high-affinity fluorescent antagonist for the ionotropic serotonin type-3 receptor.

A naphthalimide fluorophore with efficient intramolecular PET and ICT processes: application in molecular logic.

PubMed

Wang, Haixia; Wu, Haixia; Xue, Lin; Shi, Yan; Li, Xiyou

2011-08-07

A novel 4-amino-1,8-naphthalimide (NDI) with two different metal cation receptors connected at 4-amino or imide nitrogen positions respectively was designed and prepared. Significant internal charge transfer (ICT) as well as photoinduced electron transfer (PET) from the receptors to NDI is revealed by the shifted UV-vis absorption spectra and significant fluorescence quenching. Both Zn(2+) and Cu(2+) can coordinate selectively with the two cation receptors in this molecule with different affinities. The coordination of Zn(2+) with the receptor at imide nitrogen hindered the PET process and accordingly restored the quenched fluorescence of NDI. But the coordination of Zn(2+) at 4-amino position blocked the ICT process and caused significant blue-shift on the absorption peak with the fluorescence intensity unaffected. Similarly, coordination of Cu(2+) with the receptor at imide nitrogen can block the PET process, but can not restore the quenched fluorescence of compound 3 due to the paramagnetic properties of Cu(2+), which quench the fluorescence significantly instead. With Cu(2+) and Zn(2+) as two chemical inputs and absorption or fluorescence as output, several logic gate operations, such as OR, NOR and INHIBIT, can be achieved.

5-Arylvinyl-2,2′-bipyridyls: Bright “push–pull” dyes as components in fluorescent indicators for zinc ions

PubMed Central

Zhu, Lei; Younes, Ali H.; Yuan, Zhao; Clark, Ronald J.

2015-01-01

This article reviews the zinc(II)-dependent photophysical properties of arylvinylbipyridines (AVBs), a class of fluoroionophores in which 2,2′-bipyridyl and an aryl moiety are electronically conjugated. Zinc(II) binding of an AVB may lead to an emission bathochromic shift of the fluoroionophore without diminishing its fluorescence quantum yield. This observation can be explained using the excited state model of electron donor–π bridge–electron acceptor “push–pull” fluorophores, in which the bipy moiety acts as an electron acceptor, and zinc(II)-coordination strengthens its electron affinity. The spectral sensitivity of bipy-containing fluoroionophores, such as AVBs, to zinc(II) can be exploited to prepare fluorescent indicators for this ion. In several cases, AVB moieties are incorporated in fluorescent heteroditopic ligands, so that the variation of zinc(II) concentration over a relatively large range can be correlated to fluorescence changes in either intensity or color. AVB fluoroionophores are also used to introduce an intramolecular Förster resonance energy transfer (FRET) strategy for creating zinc(II) indicators with high photostability and a narrow emission band, two desired characteristics of dyes used in fluorescence microscopy. PMID:26190906

Application of fluorescently labeled tracer technique for detection of natural active macromolecules in Chinese medicine.

PubMed

Zeng, Qiao-Hui; Zhang, Xue-Wu; Xu, Kai-Peng; Jiang, Jian-Guo

2014-02-01

Active substances in traditional Chinese Medicine (TCM) contain not only a variety of small molecules, but also many other macromolecules (TCMMs), such as proteins, peptides and polysaccharides. Active TCMM can achieve good therapeutic effects by regulating the body's overall function with lower side effects. This review summarized the literatures published in recent years on the application of fluorescently labeled tracer technique for detection of natural active macromolecules in TCM. Classified by fluorescent markers, applications of fluorescein, rhodamine, and quantum dots (QDs) in TCMM active tracer are reviewed, and the methods and principles of TCMM fluorescent marker are illustrated. Studies on active TCMMs and their action mechanism are quite difficult due to a multitarget, multicomponent, and multipath system of TCM. However, the development of fluorescently labeled active tracer technique (FLATT) provides this research with new tools. Traditional fluorescent markers have many deficiencies, such as easily quenched, short luminous cycle, and intrinsic toxicity. Relatively, FLATT has many obvious advantages, and its application in TCMM is still at the early stage. In order to improve the overall level of fluorescence labeling in TCMM active tracer, the improvement on FLATT's detection sensitivity and biological affinity is urgent and critical to allow study of these interesting molecules.

A small-molecule-linked DNA-graphene oxide-based fluorescence-sensing system for detection of biotin.

PubMed

Zhang, Hao; Li, Yan; Su, Xingguang

2013-11-15

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin-DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3' to the 5' terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5-20nmol/L. The detection limit for biotin is 0.44nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein-small molecule ligand binding. Copyright © 2013 Elsevier Inc. All rights reserved.

An aptamer-based fluorescence bio-sensor for chiral recognition of arginine enantiomers

NASA Astrophysics Data System (ADS)

Yuan, Haiyan; Huang, Yunmei; Yang, Jidong; Guo, Yuan; Zeng, Xiaoqing; Zhou, Shang; Cheng, Jiawei; Zhang, Yuhui

2018-07-01

In this study, a novel aptamer - based fluorescence bio-sensor (aptamer-AuNps) was developed for chiral recognition of arginine (Arg) enantiomers based on aptamer and gold nanoparticles (AuNps). Carboxyfluorescein (FAM) labeled aptamers (Apt) were absorbed on AuNps and their fluorescence intensity could be significantly quenched by AuNps based on fluorescence resonance energy transfer (FRET). Once D-Arg or L-Arg were added into the above solution, the aptamer specifically bind to Arg enantiomers and released from AuNps, so the fluorescence intensity of D-Arg system and L-Arg system were all enhanced. The affinity of Apt to L-Arg is tighter to D-Arg, so the enhanced fluorescence signals of L-Arg system was stronger than D-Arg system. What's more, the enhanced fluorescence were directly proportional to the concentration of D-Arg and L-Arg ranging from 0-300 nM and 0-400 nM with related coefficients of 0.9939 and 0.9952, respectively. Furthermore, the method was successfully applied to detection L-Arg in human urine samples with satisfactory results. Eventually, a simple "OR" logic gate with D-Arg &L-Arg as inputs and AuNps aggregation state as outputs was fabricated, which can help us understand the chiral recognition process deeply.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

PubMed

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.

2009-09-01

A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less

MOLECULAR PROBES FOR MUSCARINIC RECEPTORS: FUNCTIONALIZED CONGENERS OF SELECTIVE MUSCARINIC ANTAGONISTS

PubMed Central

Jacobson, Kenneth A.; Fischer, Bilha; van Rhee, A. Michiel

2012-01-01

Summary The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of TAC (telenzepine amine congener), an n-decylamino derivative of the ml-selective antagonist, with the fluorescent trisulfonated pyrene dye Cascade Blue may be useful for assaying the receptor as an alternative to radiotracers. In a rat m3 receptor mutant containing a single amino acid substitution in the sixth transmembrane domain (Asn507 to Ala) the parent telenzepine lost 636-fold in affinity, while TAC lost only 27-fold. Thus, the decylamino group of TAC stabilizes the bound state and thus enhances potency by acting as a distal anchor in the receptor binding site. We have built a computer-assisted molecular model of the transmembrane regions of muscarinic receptors based on homology with the G-protein coupled receptor rhodopsin, for which a low resolution structure is known. We have coordinated the antagonist pharmacophore (tricyclic and piperazine moieties) with residues of the third and seventh helices of the rat m3 receptor. Although the decylamino chain of TAC is likely to be highly flexible and may adopt many conformations, we located one possible site for a salt bridge formation with the positively charged −NH3+ group, i.e. Asp113 in helix II. PMID:10188781

Cy5.5-labeled Affibody molecule for near-infrared fluorescent optical imaging of epidermal growth factor receptor positive tumors

NASA Astrophysics Data System (ADS)

Miao, Zheng; Ren, Gang; Liu, Hongguang; Jiang, Lei; Cheng, Zhen

2010-05-01

Affibody protein is an engineered protein scaffold with a three-helical bundle structure. Affibody molecules of small size (7 kD) have great potential for targeting overexpressed cancer biomarkers in vivo. To develop an Affibody-based molecular probe for in vivo optical imaging of epidermal growth factor receptor (EGFR) positive tumors, an anti-EGFR Affibody molecule, Ac-Cys-ZEGFR:1907 (7 kD), is site-specifically conjugated with a near-IR fluorescence dye, Cy5.5-mono-maleimide. Using fluorescent microscopy, the binding specificity of the probe Cy5.5-ZEGFR:1907 is checked by a high-EGFR-expressing A431 cell and low-EGFR-expressing MCF7 cells. The binding affinity of Cy5.5-ZEGFR:1907 (KD) to EGFR is 43.6+/-8.4 nM, as determined by flow cytometry. For an in vivo imaging study, the probe shows fast tumor targeting and good tumor contrast as early as 0.5 h postinjection (p.i.) for A431 tumors, while MCF7 tumors are barely visible. An ex vivo imaging study also demonstrates that Cy5.5-ZEGFR:1907 has high tumor, liver, and kidney uptakes at 24 h p.i.. In conclusion, Cy5.5-ZEGFR:1907 shows good affinity and high specificity to the EGFR. There is rapid achievement of good tumor-to-normal-tissue contrasts of Cy5.5-ZEGFR:1907, thus demonstrating its potential for EGFR-targeted molecular imaging of cancers.

Aza-macrocyclic Triphenylamine Ligands for G-Quadruplex Recognition.

PubMed

García-España, Enrique Victor; Pont, Isabel; González-García, Jorge; Inclán, Mario; Reynolds, Matthew; Delgado-Pinar, Estefanía; Albelda, M Teresa; Vilar, Ramon

2018-05-16

A new series of triphenylamine-based ligands with one (TPA1PY), two (TPA2PY) or three pending aza-macrocycle(s) (TPA3PY) have been synthesised and studied by means of pH-metric titrations, UV/Vis spectroscopy and fluorescence experiments. The affinity of these ligands for G-quadruplex (G4) DNA and its selectivity over duplex DNA were investigated by FRET melting assays, fluorimetric titrations and circular dichroism (CD) spectroscopy. Interestingly, the interaction of the bi- and specially the tri-branched ligand with G4 leads to a very intense red-shifted fluorescence emission band which may be associated with intermolecular aggregation between the molecule and the DNA. This light-up effect allows the application of the ligands as fluorescence probes to selectivity detect G4. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Contrasting emission behaviour of phenanthroimidazole with ZnO nanoparticles.

PubMed

Karunakaran, C; Jayabharathi, J; Sathishkumar, R; Jayamoorthy, K; Vimal, K

2013-11-01

A new fluorophore 2-(4-fluorophenyl)-1-phenyl-1H-phenanthro [9,10-d]imidazole has been synthesized and characterized by spectroscopic techniques. Nanoparticulate ZnO enhances the fluorescence of the synthesised fluorophore. The absorption, fluorescence, lifetime, cyclic voltammetry and infrared studies reveal that fluorophore is attached to the surface of ZnO semiconductor. Photo-induced electron transfer (PET) explains the enhancement of fluorescence by nanoparticulate ZnO and the apparent binding constant has been obtained. Adsorption of the fluorophore on ZnO nanoparticle lowers the HOMO and LUMO energy levels of the fluorophore. The strong adsorption of the phenanthrimidazole derivative on the surface of ZnO nanocrystals is likely due to the chemical affinity of the nitrogen atom of the organic molecule to the zinc ion on the surface of nanocrystal. Copyright © 2013 Elsevier B.V. All rights reserved.

Microwave Assisted Synthesis, Physicochemical, Photophysical, Single Crystal X-ray and DFT Studies of Novel Push-Pull Chromophores.

PubMed

Khan, Salman A; Asiri, Abdullah M; Basisi, Hadi Mussa; Arshad, Muhammad Nadeem; Sharma, Kamlesh

2015-11-01

Two push-pull chromophores were synthesized by knoevenagel condensation under microwave irradiation. The structure of synthesized chromophores were established by spectroscopic (FT-IR, (1)H NMR, (13)C NMR, EI-MS) and elemental analysis. Structure of the chromophores was further conformed by X-ray crystallographic. UV-Vis and fluorescence spectroscopy measurements provided that chromophores were good absorbent and fluorescent properties. Fluorescence polarity studies demonstrated that chromophores were sensitive to the polarity of the microenvironment provided by different solvents. Physicochemical parameters, including singlet absorption, extinction coefficient, stokes shift, oscillator strength, dipole moment and flurescence quantum yield were investigated in order to explore the analytical potential of the synthesized chromophores. In addition, the total energy, frontier molecular orbitals, hardness, electron affinity, ionization energy, electrostatic potential map were also studied computationally by using density functional theoretical method.

BODIPY-Based Fluorescent Probes for Sensing Protein Surface-Hydrophobicity.

PubMed

Dorh, Nethaniah; Zhu, Shilei; Dhungana, Kamal B; Pati, Ranjit; Luo, Fen-Tair; Liu, Haiying; Tiwari, Ashutosh

2015-12-18

Mapping surface hydrophobic interactions in proteins is key to understanding molecular recognition, biological functions, and is central to many protein misfolding diseases. Herein, we report synthesis and application of new BODIPY-based hydrophobic sensors (HPsensors) that are stable and highly fluorescent for pH values ranging from 7.0 to 9.0. Surface hydrophobic measurements of proteins (BSA, apomyoglobin, and myoglobin) by these HPsensors display much stronger signal compared to 8-anilino-1-naphthalene sulfonic acid (ANS), a commonly used hydrophobic probe; HPsensors show a 10- to 60-fold increase in signal strength for the BSA protein with affinity in the nanomolar range. This suggests that these HPsensors can be used as a sensitive indicator of protein surface hydrophobicity. A first principle approach is used to identify the molecular level mechanism for the substantial increase in the fluorescence signal strength. Our results show that conformational change and increased molecular rigidity of the dye due to its hydrophobic interaction with protein lead to fluorescence enhancement.

Live imaging of apoptotic cells in zebrafish

PubMed Central

van Ham, Tjakko J.; Mapes, James; Kokel, David; Peterson, Randall T.

2010-01-01

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.—Van Ham, T. J., Mapes, J., Kokel, D., Peterson, R. T. Live imaging of apoptotic cells in zebrafish. PMID:20601526

To Your Health: NLM update transcript - Breast cancer treatment sans chemotherapy?

MedlinePlus

... transcript061118.html To Your Health: NLM update Transcript Breast cancer treatment sans chemotherapy? : 06/11/2018 To use ... 50 with a midrange or modest risk of breast cancer safely may skip chemotherapy as part of their ...

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

PubMed Central

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods. PMID:28255355

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

PubMed Central

Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

2012-01-01

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

Contract W911NF-07-1-0139 (Massachusetts Institute of Technology)

DTIC Science & Technology

2013-07-09

materials were effective in nucleophilic degradation of organophosphorous (OP) esters represented by CWA simulants and pesticides. Studies demonstrated...separate the cells effectively from suspension using a magnet. A fluorescence dye displacement assay shows strong affinities of the nanoparticles...as other imidazole groups, in its proximity. In that regard, we were interested in potential neighboring effects of the imidazole groups concentrated

Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15.

PubMed

Liu, Mengjie; Richardson, Rachel R; Mountford, Simon J; Zhang, Lei; Tempone, Matheus H; Herzog, Herbert; Holliday, Nicholas D; Thompson, Philip E

2016-09-21

Traceable truncated Neuropeptide Y (NPY) analogues with Y1 receptor (Y1R) affinity and selectivity are highly desirable tools in studying receptor location, regulation, and biological functions. A range of fluorescently labeled analogues of a reported Y1R/Y4R preferring ligand BVD-15 have been prepared and evaluated using high content imaging techniques. One peptide, [Lys(2)(sCy5), Arg(4)]BVD-15, was characterized as an Y1R antagonist with a pKD of 7.2 measured by saturation analysis using fluorescent imaging. The peptide showed 8-fold lower affinity for Y4R (pKD = 6.2) and was a partial agonist at this receptor. The suitability of [Lys(2)(sCy5), Arg(4)]BVD-15 for Y1R and Y4R competition binding experiments was also demonstrated in intact cells. The nature of the label was shown to be critical with replacement of sCy5 by the more hydrophobic Cy5.5 resulting in a switch from Y1R antagonist to Y1R partial agonist.

Spectroscopic and molecular modeling approaches to investigate the interaction of bisphenol A, bisphenol F and their diglycidyl ethers with PPARα.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Ruan, Ping; Ren, Dayong; Dai, Weichang; Yu, Hansong; Li, Tiezhu

2017-08-01

A fluorescence polarization (FP) assay for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF), bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) was developed. The method was based on the competition between bisphenols (BPs) and fluorescein-labeled dexamethasone derivative (Dex-fl) for mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD). A recombinant soluble protein derivative mPPARα-LBD* was prepared, then in vitro binding of 4 BPs to mPPARα-LBD* was investigated. Fluorescence polarization assay showed that these compounds exhibited different binding potencies with mPPARα-LBD*. Additionally, molecular dynamics simulations were performed to further understand the mechanism of BPs binding affinity for mPPARα-LBD*. Docking results elucidated that the driving forces for the binding of BPs to mPPARα-LBD* were predominantly dependent on hydrophobic and hydrogen-bonding interactions. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.7258). The proposed method has potential for multi-residue detection of BPA, BPF, BADGE, and BFDGE. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preliminary selection and evaluation of the binding of aptamers against a Hantavirus antigen using fluorescence spectroscopy and modeling

NASA Astrophysics Data System (ADS)

Missailidis, Sotiris; de Oliveira, Renata Carvalho; Silva, Dilson; Cortez, Célia Martins; Guterres, Alexandro; Vicente, Luciana Helena Bassan; de Godoy, Daniela Tupy; Lemos, Elba

2015-12-01

In this study we have aimed to develop novel aptamers against the Hantavirus nucleoprotein N, a valid antigen already used in the Hantavirus reference laboratory of the Institute Oswaldo Cruz in Rio de Janeiro, Brazil. Such aptamers, if they are found to bind with high affinity and specificity for the selected hantavirus antigen, they could be translated into novel diagnostic assays with the ability to provide early detection for hantaviroses and their related disease syndromes. In a preliminary screening, we have managed to identify three aptamer species. We have analyzed a short and a long version of these aptamer using fluorescence spectroscopy and modelled their binding. We have identified Stern-Volmer constants for the selected aptamers, which have shown affinity for their target, with a different binding between the short and the long versions of them. Short aptamers have shown to have a higher Stern-Volmer constant and the ability to potentially bind to more than one binding site on the antigen. The information provided by the spectroscopic screening has been invaluable in allowing us to define candidates for further development into diagnostic assays.

A novel fluorescent aptasensor for the highly sensitive and selective detection of cardiac troponin I based on a graphene oxide platform.

PubMed

Liu, Dongkui; Lu, Xing; Yang, Yiwen; Zhai, Yunyun; Zhang, Jian; Li, Lei

2018-05-04

Acute myocardial infarction (AMI) is one of the leading risks to global health. Thus, the rapid, accurate early diagnosis of AMI is highly critical. Human cardiac troponin I (cTnI) has been regarded as a golden biomarker for AMI due to its excellent selectivity. In this work, a novel fluorescent aptasensor based on a graphene oxide (GO) platform was developed for the highly sensitive and selective detection of cTnI. GO binds to the fluorescent anti-cTnI aptamer and quenches its fluorescence. In the presence of cTnI, the fluorescent anti-cTnI aptamer leaves the surface of GO, combines with cTnI because of the powerful affinity of the fluorescent anti-cTnI aptamer and cTnI, and then restores the fluorescence of the fluorescent anti-cTnI aptamer. Fluorescence-enhanced detection is highly sensitive and selective to cTnI. The method exhibited good analytical performance with a reasonable dynamic linearity at the concentration range of 0.10-6.0 ng/mL and a low detection limit of 0.07 ng/mL (S/N = 3). The fluorescent aptasensor also exhibited high selectivity toward cTnI compared with other interference proteins. The proposed method may be a potentially useful tool for cTnI determination in human serum. Graphical abstract A novel fluorescent aptasensor for the highly sensitive and selective detection of cardiac troponin I based on a graphene oxide platform.

Changes in small angle X-ray scattering parameters observed upon ligand binding to rabbit muscle pyruvate kinase are not correlated with allosteric transitions†

PubMed Central

Fenton, Aron W.; Williams, Rachel; Trewhella, Jill

2010-01-01

Protein fluorescence and small-angle X-ray scattering (SAXS) have been used to monitor effector affinity and conformational changes previously associated with allosteric regulation in rabbit muscle pyruvate kinase (M1-PYK). In the absence of substrate (phosphoenolpyruvate; PEP), SAXS-monitored conformational changes in M1-PYK elicited by the binding of phenylalanine (an allosteric inhibitor that reduces the affinity of M1-PYK for PEP) are similar to those observed upon binding of alanine or 2-aminobutyric acid. Under the current assay conditions, these small amino acids bind to the protein, but elicit a minimal change in the affinity of the protein for PEP. Therefore, if changes in scattering signatures represent cleft closure via domain rotation as previously interpreted, it can be concluded that these motions are not sufficient to elicit allosteric inhibition. Additionally, although PEP has similar affinities for the free enzyme and the M1-PYK/small-amino-acid complexes (i.e. the small amino acids have minimal allosteric effects), PEP binding elicits different changes in the SAXS signature of the free enzyme vs. the M1-PYK/small-amino-acid complexes. PMID:20712377

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content.

PubMed

Benítez, Sonia; Villegas, Virtudes; Bancells, Cristina; Jorba, Oscar; González-Sastre, Francesc; Ordóñez-Llanos, Jordi; Sánchez-Quesada, José Luis

2004-12-21

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

PubMed

Besbrugge, Nienke; Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; Kulkarni, Shubhada R; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Bontinck, Michiel; Aesaert, Stijn; Impens, Francis; Gevaert, Kris; Van Damme, Daniel; Van Lijsebettens, Mieke; Inzé, Dirk; Vandepoele, Klaas; Nelissen, Hilde; De Jaeger, Geert

2018-06-01

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS yellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS yellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS yellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research. © 2018 American Society of Plant Biologists. All rights reserved.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components

PubMed Central

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, UA

2014-01-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and Impact of the Study Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. PMID:24935714

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

PubMed

Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

2003-06-24

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

Affimer proteins are versatile and renewable affinity reagents

PubMed Central

Tiede, Christian; Bedford, Robert; Heseltine, Sophie J; Smith, Gina; Wijetunga, Imeshi; Ross, Rebecca; AlQallaf, Danah; Roberts, Ashley PE; Balls, Alexander; Curd, Alistair; Hughes, Ruth E; Martin, Heather; Needham, Sarah R; Zanetti-Domingues, Laura C; Sadigh, Yashar; Peacock, Thomas P; Tang, Anna A; Gibson, Naomi; Kyle, Hannah; Platt, Geoffrey W; Ingram, Nicola; Taylor, Thomas; Coletta, Louise P; Manfield, Iain; Knowles, Margaret; Bell, Sandra; Esteves, Filomena; Maqbool, Azhar; Prasad, Raj K; Drinkhill, Mark; Bon, Robin S; Patel, Vikesh; Goodchild, Sarah A; Martin-Fernandez, Marisa; Owens, Ray J; Nettleship, Joanne E; Webb, Michael E; Harrison, Michael; Lippiat, Jonathan D; Ponnambalam, Sreenivasan; Peckham, Michelle; Smith, Alastair; Ferrigno, Paul Ko; Johnson, Matt; McPherson, Michael J; Tomlinson, Darren Charles

2017-01-01

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001 PMID:28654419

Surface-modified multifunctional MIP nanoparticles

PubMed Central

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; de Vargas Sansalvador, Isabel Perez; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J.; Piletsky, Sergey

2015-01-01

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly-sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinyl ferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors. PMID:23503559

Rationale, Design, and Methodology of the APOLLON trial: A comPrehensive, ObservationaL registry of heart faiLure with midrange and preserved ejectiON fraction.

PubMed

Özlek, Bülent; Özlek, Eda; Çelik, Oğuzhan; Çil, Cem; Doğan, Volkan; Tekinalp, Mehmet; Zencirkıran Ağuş, Hicaz; Kahraman, Serkan; Ösken, Altuğ; Rencüzoğulları, İbrahim; Tanık, Veysel Ozan; Bekar, Lütfü; Çakır, Mustafa Ozan; Kaya, Bedri Caner; Tibilli, Hakan; Çelik, Yunus; Başaran, Özcan; Mert, Kadir Uğur; Sevinç, Samet; Demirci, Erkan; Dondurmacı, Engin; Biteker, Murat

2018-05-01

Although almost half of chronic heart failure (HF) patients have mid-range (HFmrEF) and preserved left-ventricular ejection fraction (HFpEF), no studies have been carried out with these patients in our country. This study aims to determine the demographic characteristics and current status of the clinical background of HFmrEF and HFpEF patients in a multicenter trial. A comPrehensive, ObservationaL registry of heart faiLure with mid range and preserved ejectiON fraction (APOLLON) trial will be an observational, multicenter, and noninterventional study conducted in Turkey. The study population will include 1065 patients from 12 sites in Turkey. All data will be collected at one point in time and the current clinical practice will be evaluated (ClinicalTrials.gov number NCT03026114). We will enroll all consecutive patients admitted to the cardiology clinics who were at least 18 years of age and had New York Heart Association class II, III, or IV HF, elevated brain natriuretic peptide levels within the last 30 days, and an left ventricular ejection fraction (LVEF) of at least 40%. Patients fulfilling the exclusion criteria will not be included in the study. Patients will be stratified into two categories according to LVEF: mid-range EF (HFmrEF, LVEF 40%-49%) and preserved EF (HFpEF, LVEF ≥50%). Regional quota sampling will be performed to ensure that the sample was representative of the Turkish population. Demographic, lifestyle, medical, and therapeutic data will be collected by this specific survey. The APOLLON trial will be the largest and most comprehensive study in Turkey evaluating HF patients with a LVEF ≥40% and will also be the first study to specifically analyze the recently designated HFmrEF category.

Genomic mid-range inhomogeneity correlates with an abundance of RNA secondary structures

PubMed Central

Bechtel, Jason M; Wittenschlaeger, Thomas; Dwyer, Trisha; Song, Jun; Arunachalam, Sasi; Ramakrishnan, Sadeesh K; Shepard, Samuel; Fedorov, Alexei

2008-01-01

Background Genomes possess different levels of non-randomness, in particular, an inhomogeneity in their nucleotide composition. Inhomogeneity is manifest from the short-range where neighboring nucleotides influence the choice of base at a site, to the long-range, commonly known as isochores, where a particular base composition can span millions of nucleotides. A separate genomic issue that has yet to be thoroughly elucidated is the role that RNA secondary structure (SS) plays in gene expression. Results We present novel data and approaches that show that a mid-range inhomogeneity (~30 to 1000 nt) not only exists in mammalian genomes but is also significantly associated with strong RNA SS. A whole-genome bioinformatics investigation of local SS in a set of 11,315 non-redundant human pre-mRNA sequences has been carried out. Four distinct components of these molecules (5'-UTRs, exons, introns and 3'-UTRs) were considered separately, since they differ in overall nucleotide composition, sequence motifs and periodicities. For each pre-mRNA component, the abundance of strong local SS (< -25 kcal/mol) was a factor of two to ten greater than a random expectation model. The randomization process preserves the short-range inhomogeneity of the corresponding natural sequences, thus, eliminating short-range signals as possible contributors to any observed phenomena. Conclusion We demonstrate that the excess of strong local SS in pre-mRNAs is linked to the little explored phenomenon of genomic mid-range inhomogeneity (MRI). MRI is an interdependence between nucleotide choice and base composition over a distance of 20–1000 nt. Additionally, we have created a public computational resource to support further study of genomic MRI. PMID:18549495

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Electrochemical affinity biosensors for detection of mycotoxins: A review.

PubMed

Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

2013-11-15

This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

Fluorescent detection of apoptotic cells using a family of zinc coordination complexes with selective affinity for membrane surfaces that are enriched with phosphatidylserine.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Bradley D.; Lambert, Timothy N.; Lakshmi, C.

2005-03-01

The appearance of phosphatidylserine on the membrane surface of apoptotic cells (Jurkat, CHO, HeLa) is monitored by using a family of bis(Zn{sup 2+}-2,2{prime}-dipicolylamine) coordination compounds with appended fluorescein or biotin groups as reporter elements. The phosphatidylserine affinity group is also conjugated directly to a CdSe/CdS quantum dot to produce a probe suitable for prolonged observation without photobleaching. Apoptosis can be detected under a wide variety of conditions, including variations in temperature, incubation time, and binding media. Binding of each probe appears to be restricted to the cell membrane exterior, because no staining of organelles or internal membranes is observed.

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

PubMed

Sueck, Franziska; Poór, Miklós; Faisal, Zelma; Gertzen, Christoph G W; Cramer, Benedikt; Lemli, Beáta; Kunsági-Máté, Sándor; Gohlke, Holger; Humpf, Hans-Ulrich

2018-06-22

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus . 2′ R -Ochratoxin A (2′ R -OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′ R -OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′ R -OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′ R -OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′ R -OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a log K of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′ R -OTA, a lower, but still high, log K of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′ R -OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′ R -OTA found in human blood samples.

Personality Traits and Family Styles of Combat Medics in Training.

PubMed

Escolas, Hollie D; Ray, Lashawnna N; Escolas, Sandra M

2016-06-01

This descriptive study examines the relationship between four family types and five personality traits. The four family types are balanced, moderately balanced, midrange, and extreme. The five personality traits are extraversion, openness to experiences, agreeableness, emotional stability, and conscientiousness. Data were collected through anonymous questionnaires distributed to combat-naïve Soldiers at the beginning of their advanced individual training. This study utilized the Family Adaptability and Cohesion Evaluation Scale1 and the Ten-Item Personality Inventory2 as measures. Overall the analyses found that participants classified as a balanced family type scored significantly higher on the personality traits of extraversion, agreeableness, and openness to experience than those classified in the family types of extreme, midrange, and moderately balanced. It appears that family types are associated with personality traits. This study opens doors to future research including looking at how family and personality types relate to each other in military units and personnel. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

On the modeling of irradiation-induced homogeneous precipitation in proton-bombarded Ni-Si solid solutions

NASA Astrophysics Data System (ADS)

Lam, Nghi Q.; Janghorban, K.; Ardell, A. J.

1981-10-01

Irradiation-induced solute redistribution leading to precipitation of coherent γ' particles in undersaturated Ni-based solid solutions containing 6 and 8 at.% Si during 400-keV proton bombardment was modeled, based on the concept of solute segregation in concentrated alloys under spatially-dependent defect production conditions. The combined effects of (i) an extremely large difference between the defect production rates in the peak-damage and mid-range regions during irradiation and (ii) a preferential coupling between the interstitial and solute fluxes generate a net transient flux of Si atoms into the mid-range region, which is much larger than the solute flux out of this location. As a result, the Si concentration exceeds the solubility limit and homogeneous precipitation of the γ' phase occurs in this particular region of the irradiated samples. The spatial, compositional and temperature dependences of irradiation-induced homogeneous precipitation derived from the present theoretical calculations are in good qualitative agreement with experimental observations

Bioconjugated Quantum Dots for In Vivo Molecular and Cellular Imaging

PubMed Central

Smith, Andrew M.; Duan, Hongwei; Mohs, Aaron M.; Nie, Shuming

2008-01-01

Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology. PMID:18495291

Probing the binding interaction of a phenazinium dye with serum transport proteins: a combined fluorometric and circular dichroism study.

PubMed

Bose, Debosreeta; Sarkar, Deboleena; Chattopadhyay, Nitin

2010-01-01

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of alpha-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.

Synthesis and cell imaging applications of fluorescent mono/di/tri-heterocyclyl-2,6-dicyanoanilines.

PubMed

Pisal, Mahesh M; Annadate, Ritesh A; Athalye, Meghana C; Kumar, Deepak; Chavan, Subhash P; Sarkar, Dhiman; Borate, Hanumant B

2017-02-15

Synthesis of 3,4,5-triheterocyclyl-2,6-dicyanoanilines, starting from heterocyclic aldehydes and 1,2-diheterocycle-substituted ethanones, is described. 2,6-Dicyanoanilines with one or two heterocyclic substituents have also been synthesized. It was found that some of these molecules have selective cell-staining properties useful for cell imaging applications. The compounds 1g, 10f and 11 were found to stain cytoplasm of the cells in contact but not the nucleus while the compound 12 showed affinity to apoptotic cells resulting in blue fluorescence. The cell imaging results with compound 12 were similar to Annexin V-FITC, a known reagent containing recombinant Annexin V conjugated to green-fluorescent FITC dye, used for detection of apoptotic cells. These compounds were found to be non-cytotoxic and have potential application as cell imaging agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

PubMed

Biela, Ewa; Galas, Jerzy; Lee, Brian; Johnson, Gary L; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2013-06-01

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials. Copyright © 2013 International Society for Advancement of Cytometry.

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

PubMed

Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

2005-07-19

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

Cu2 + modulated nitrogen-doped grapheme quantum dots as a turn-off/on fluorescence sensor for the selective detection of histidine in biological fluid

NASA Astrophysics Data System (ADS)

Wang, Zhiyu; Fan, ZheFeng

2018-01-01

A highly sensitive sensor for detection of histidine (His) based on the nitrogen-doped graphene quantum dots (N-GQDs)-Cu2 + system has been designed. The N-GQDs were synthesized by one-step hydrothermal approach according to previous report. The fluorescence of N-GQDs can be effectively quenched by Cu2 + due to the binding between Cu2 + and functional groups on the surface of N-GQDs. The high affinity of His to Cu2 + enables Cu2 + to be dissociated from the surface of N-GQDs and recovering the fluorescence. The sensor displayed a sensitive response to His in the concentration range of 0-35 μmol L- 1, with a detection limit of 72.2 nmol L- 1. The proposed method is successfully applied to detect His in samples with a recovery range of 96-102%.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

PubMed

Okamatsu, Masatoshi; Feng, Fei; Ohyanagi, Tatsuya; Nagahori, Noriko; Someya, Kazuhiko; Sakoda, Yoshihiro; Miura, Nobuaki; Nishimura, Shin-Ichiro; Kida, Hiroshi

2013-02-01

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. Copyright © 2012 Elsevier B.V. All rights reserved.

Polydopamine Nanotubes as an Effective Fluorescent Quencher for Highly Sensitive and Selective Detection of Biomolecules Assisted with Exonuclease III Amplification.

PubMed

Fan, Daoqing; Zhu, Xiaoqing; Zhai, Qingfeng; Wang, Erkang; Dong, Shaojun

2016-09-20

In this work, the effective fluorescence quenching ability of polydopamine nanotubes (PDANTs) toward various fluorescent dyes was studied and further applied to fluorescent biosensing for the first time. The PDANTs could quench the fluorophores with different emission frequencies, aminomethylcoumarin acetate (AMCA), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), and Cy5. All the quenching efficiencies reached to more than 97%. Taking advantage of PDANTs' different affinities toward ssDNA and dsDNA and utilizing the complex of FAM-labeled ssDNA and PDANTs as a sensing platform, we achieved highly sensitive and selective detection of human immunodeficiency virus (HIV) DNA and adenosine triphosphate (ATP) assisted with Exonuclease III amplification. The limits of detection (LODs) of HIV DNA and ATP reached to 3.5 pM and 150 nM, respectively, which were all lower than that of previous nanoquenchers with Exo III amplification, and the platform also presented good applicability in biological samples. Fluorescent sensing applications of this nanotube enlightened other targets detection based upon it and enriched the building blocks of fluorescent sensing platforms. This polydopamine nanotube also possesses excellent biocompatibility and biodegradability, which is suitable for future drug delivery, cell imaging, and other biological applications.

A novel fluorescent aptasensor based on gold and silica nanoparticles for the ultrasensitive detection of ochratoxin A

NASA Astrophysics Data System (ADS)

Taghdisi, Seyed Mohammad; Danesh, Noor Mohammad; Beheshti, Hamed Reza; Ramezani, Mohammad; Abnous, Khalil

2016-02-01

Analytical approaches for the detection and quantitation of ochratoxin A (OTA) in blood serum and food products are high in demand. In this study, a fluorescent aptamer-based sensor (aptasensor) is developed for the selective and sensitive detection of OTA, based on a complementary strand of aptamer (CS) and two types of nanoparticles, gold nanoparticles (AuNPs) and silica nanoparticles (SNPs) coated with streptavidin. The fabricated aptasensor inherits the characteristics of SNPs, as enhancers of fluorescence intensity; AuNPs, such as large surface area and unique optical properties; and high affinity of the aptamer toward its target compared to its CS. In the absence of OTA, no FAM and biotin-labeled CS is in the environment of the SNPs coated with streptavidin, which leads to no fluorescence emission. In the presence of the target, an FAM and biotin-labeled CS-SNPs coated with streptavidin conjugate is formed, thus resulting in a very strong fluorescence emission. The designed fluorescent aptasensor exhibits high selectivity toward OTA with a limit of detection (LOD) as low as 0.098 nM. Furthermore, the fabricated aptasensor was successfully applied for the detection of OTA in grape juice and serum with LODs of 0.113 and 0.152 nM, respectively.

Facile synthesis, cytotoxicity and bioimaging of Fe(3+) selective fluorescent chemosensor.

PubMed

Saleem, Muhammad; Abdullah, Razack; Ali, Anser; Park, Bong Joo; Choi, Eun Ha; Hong, In Seok; Lee, Ki Hwan

2014-04-01

The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe(3+) with micro molar concentration level. Through fluorescence titration at 599nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe(3+) while it appeared no emission in case of the competitive ions (Sc(3+), Yb(3+), In(3+), Ce(3+), Sm(3+), Cr(3+), Sn(2+), Pb(2+), Ni(2+), Co(2+), Cu(2+), Ba(2+), Ca(2+), Mg(2+), Ag(+), Cs(+), Cu(+), K(+)) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand-iron complex solution was quenched by addition of KCN which snatches the Fe(3+) from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fluorescent 2D WS2 Nanosheets Bearing Chemical Affinity Elements for the Recognition of Glycated Hemoglobin.

PubMed

Yang, Jin-Kyoung; Lee, Hye-Rim; Hwang, In-Jun; Kim, Hye-In; Yim, DaBin; Kim, Jong-Ho

2018-05-14

It is required to exfoliate and functionalize 2D transition metal dichalcogenides (TMDs) in an aqueous solution for biological and medical applications. Herein, the approach for the simultaneous exfoliation and functionalization of 2D WS 2 nanosheets using boronic acid-modified poly(vinyl alcohol) (B-PVA) in an aqueous solution is reported, and the B-PVA-functionalized WS 2 nanosheets (B-PVA-WS 2 ) are exploited as a fluorescent biosensor for the detection of glycated hemoglobin, HbA1c. The synthetic B-PVA polymer facilitates the exfoliation and functionalization of WS 2 nanosheets from the bulk counterpart in the aqueous solution via a pulsed sonication process, resulting in fluorescent B-PVA-WS 2 nanohybrids with a specific recognition of HbA1c. The fluorescence of the B-PVA-WS 2 is quenched in the presence of HbA1c, whereas PVA-functionalized WS 2 (PVA-WS 2 ), not bearing boronic acid as a recognition moiety, shows no fluorescence changes upon the addition of the target. The B-PVA-WS 2 is able to selectively detect HbA1c at the concentration as low as 3.3 × 10 -8 m based on its specific fluorescence quenching. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein sorption on polymer surfaces measured by fluorescence labels.

PubMed

Brynda, E; Drobník, J; Vacík, J; Kálal, J

1978-01-01

Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about 10(-8) g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.

Fluorescence resonance energy transfer between green fluorescent protein and doxorubicin enabled by DNA nanotechnology.

PubMed

Heger, Zbynek; Kominkova, Marketa; Cernei, Natalia; Krejcova, Ludmila; Kopel, Pavel; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

2014-12-01

DNA nanotechnology is a rapidly growing research area, where DNA may be used for wide range of applications such as construction of nanodevices serving for large scale of diverse purposes. Likewise a panel of various purified fluorescent proteins is investigated for their ability to emit their typical fluorescence spectra under influence of particular excitation. Hence these proteins may form ideal donor molecules for assembly of fluorescence resonance emission transfer (FRET) constructions. To extend the application possibilities of fluorescent proteins, while using DNA nanotechnology, we developed nanoconstruction comprising green fluorescent protein (GFP) bound onto surface of surface active nanomaghemite and functionalized with gold nanoparticles. We took advantage of natural affinity between gold and thiol moieties, which were modified to bind DNA fragment. Finally we enclosed doxorubicin into fullerene cages. Doxorubicin intercalated in DNA fragment bound on the particles and thus we were able to connect these parts together. Because GFP behaved as a donor and doxorubicin as an acceptor using excitation wavelength for GFP (395 nm) in emission wavelength of doxorubicin (590 nm) FRET was observed. This nanoconstruction may serve as a double-labeled transporter of doxorubicin guided by force of external magnetic force owing to the presence of nanomaghemite. Further nanomaghemite offers the possibility of using this technology for thermotherapy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

PubMed

Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

An aptamer-based fluorescence bio-sensor for chiral recognition of arginine enantiomers.

PubMed

Yuan, Haiyan; Huang, Yunmei; Yang, Jidong; Guo, Yuan; Zeng, Xiaoqing; Zhou, Shang; Cheng, Jiawei; Zhang, Yuhui

2018-07-05

In this study, a novel aptamer - based fluorescence bio-sensor (aptamer-AuNps) was developed for chiral recognition of arginine (Arg) enantiomers based on aptamer and gold nanoparticles (AuNps). Carboxyfluorescein (FAM) labeled aptamers (Apt) were absorbed on AuNps and their fluorescence intensity could be significantly quenched by AuNps based on fluorescence resonance energy transfer (FRET). Once d-Arg or l-Arg were added into the above solution, the aptamer specifically bind to Arg enantiomers and released from AuNps, so the fluorescence intensity of d-Arg system and l-Arg system were all enhanced. The affinity of Apt to l-Arg is tighter to d-Arg, so the enhanced fluorescence signals of l-Arg system was stronger than d-Arg system. What's more, the enhanced fluorescence were directly proportional to the concentration of d-Arg and l-Arg ranging from 0-300 nM and 0-400 nM with related coefficients of 0.9939 and 0.9952, respectively. Furthermore, the method was successfully applied to detection l-Arg in human urine samples with satisfactory results. Eventually, a simple "OR" logic gate with d-Arg &l-Arg as inputs and AuNps aggregation state as outputs was fabricated, which can help us understand the chiral recognition process deeply. Copyright © 2018 Elsevier B.V. All rights reserved.

Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry.

PubMed

Morell, Montse; Espargaro, Alba; Aviles, Francesc Xavier; Ventura, Salvador

2008-01-01

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells.

PubMed

Zhang, Jingtuo; Li, Cong; Dutta, Colina; Fang, Mingxi; Zhang, Shuwei; Tiwari, Ashutosh; Werner, Thomas; Luo, Fen-Tair; Liu, Haiying

2017-05-22

A novel near-infrared fluorescent probe for β-galactosidase has been developed based on a hemicyanine skeleton, which is conjugated with a d-galactose residue via a glycosidic bond. The probe serves as a substrate of β-galactosidase and displays rapid and sensitive turn-on fluorescent responses to β-galactosidase in aqueous solution. A 12.8-fold enhancement of fluorescence intensity at 703 nm was observed after incubation of 10 nM of β-galactosidase with 5 μM probe for 10 min. The probe can sensitively detect as little as 0.1 nM of β-galactosidase and shows linear responses to the enzyme concentration below 1.4 nM. The kinetic study showed that the probe has high binding affinity to β-galactosidase with K m  = 3.6 μM. The probe was used to detect β-galactosidase in living cells by employing the premature cell senescence model. The probe exhibited strong fluorescent signals in senescent cells but not in normal cells, which demonstrates that the probe is able to detect the endogenous senescence-associated β-galactosidase in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescent indicator dyes for calcium ions

NASA Technical Reports Server (NTRS)

Grynkiewicz, Grzegorz (Inventor); Tsien, Roger Y. (Inventor)

1986-01-01

The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, quin2, the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues. The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.

Smart coumarin-tagged imprinted polymers for the rapid detection of tamoxifen.

PubMed

Ray, Judith V; Mirata, Fosca; Pérollier, Celine; Arotcarena, Michel; Bayoudh, Sami; Resmini, Marina

2016-03-01

A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 μm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.

Measuring Equilibrium Binding Affinity of Biological Macromolecules in Solution by Thermophoresis

DTIC Science & Technology

2015-05-18

SECURITY CLASSIFICATION OF: The primary research focus of the San Diego State University (SDSU) Structural Biochemistry Laboratory led by Dr. Tom...U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 Biochemistry , Biophysics, Fluorescence, IkappaB Kinase...University (SDSU) Structural Biochemistry Laboratory led by Dr. Tom Huxford is to understand how the assembly of macromolecular complexes gives rise

Submicrometer Metallic Barcodes

NASA Astrophysics Data System (ADS)

Nicewarner-Peña, Sheila R.; Freeman, R. Griffith; Reiss, Brian D.; He, Lin; Peña, David J.; Walton, Ian D.; Cromer, Remy; Keating, Christine D.; Natan, Michael J.

2001-10-01

We synthesized multimetal microrods intrinsically encoded with submicrometer stripes. Complex striping patterns are readily prepared by sequential electrochemical deposition of metal ions into templates with uniformly sized pores. The differential reflectivity of adjacent stripes enables identification of the striping patterns by conventional light microscopy. This readout mechanism does not interfere with the use of fluorescence for detection of analytes bound to particles by affinity capture, as demonstrated by DNA and protein bioassays.

A human pilot study of the fluorescence affinity sensor for continuous glucose monitoring in diabetes.

PubMed

Dutt-Ballerstadt, Ralph; Evans, Colton; Pillai, Arun P; Orzeck, Eric; Drabek, Rafal; Gowda, Ashok; McNichols, Roger

2012-03-01

We report results of a pilot clinical study of a subcutaneous fluorescence affinity sensor (FAS) for continuous glucose monitoring conducted in people with type 1 and type 2 diabetes. The device was assessed based on performance, safety, and comfort level under acute conditions (4 h). A second-generation FAS (BioTex Inc., Houston, TX) was subcutaneously implanted in the abdomens of 12 people with diabetes, and its acute performance to excursions in blood glucose was monitored over 4 h. After 30-60 min the subjects, who all had fasting blood glucose levels of less than 200 mg/dl, received a glucose bolus of 75 g/liter dextrose by oral administration. Capillary blood glucose samples were obtained from the finger tip. The FAS data were retrospectively evaluated by linear least squares regression analysis and by the Clarke error grid method. Comfort levels during insertion, operation, and sensor removal were scored by the subjects using an analog pain scale. After retrospective calibration of 17 sensors implanted in 12 subjects, error grid analysis showed 97% of the paired values in zones A and B and 1.5% in zones C and D, respectively. The mean absolute relative error between sensor signal and capillary blood glucose was 13% [±15% standard deviation (SD), 100-350 mg/dl] with an average correlation coefficient of 0.84 (±0.24 SD). The actual average "warm-up" time for the FAS readings, at which highest correlation with glucose readings was determined, was 65 (±32 SD) min. Mean time lag was 4 (±5 SD) min during the initial operational hours. Pain levels during insertion and operation were modest. The in vivo performance of the FAS demonstrates feasibility of the fluorescence affinity technology to determine blood glucose excursions accurately and safely under acute dynamic conditions in humans with type 1 and type 2 diabetes. Specific engineering challenges to sensor and instrumentation robustness remain. Further studies will be required to validate its promising performance over longer implantation duration (5-7 days) in people with diabetes. © 2012 Diabetes Technology Society.

Identification, Expression Profiling and Fluorescence-Based Binding Assays of a Chemosensory Protein Gene from the Western Flower Thrips, Frankliniella occidentalis

PubMed Central

Zhang, Zhi-Ke; Lei, Zhong-Ren

2015-01-01

Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four—cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating the development of the first instar nymph and mediate F. occidentalis host recognition. PMID:25635391

Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella occidentalis.

PubMed

Zhang, Zhi-Ke; Lei, Zhong-Ren

2015-01-01

Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four-cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating the development of the first instar nymph and mediate F. occidentalis host recognition.

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

PubMed

Rohrback, Suzanne E; Wheatly, Michele G; Gillen, Christopher M

2015-01-01

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP. Copyright © 2014 Elsevier Inc. All rights reserved.

Selective binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid to peroxisome proliferator-activated receptor gamma allows ligand identification and characterization.

PubMed

Zorrilla, Silvia; Garzón, Beatriz; Pérez-Sala, Dolores

2010-04-01

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation, and carcinogenesis. PPARgamma transcriptional activity is modulated by specific ligands that promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARgamma-LBD (ligand binding domain), displaying negligible interaction with other nuclear receptors such as PPARalpha and retinoid X receptor alpha (RXRalpha). ANS binding is competed by PPARgamma agonists such as rosiglitazone, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), and 9,10-dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) (CAY10410). Moreover, the affinity of PPARgamma for these ligands, determined through ANS competition titrations, is within the range of that reported previously, thereby suggesting that ANS competition could be useful in the screening and characterization of novel PPARgamma agonists. In contrast, gel-based competition assays showed limited performance with noncovalently bound ligands. We applied the ANS binding assay to characterize a biotinylated analog of 15d-PGJ(2) that does not activate PPAR in cells. We found that although this compound bound to PPARgamma with low affinity, it failed to promote PPARgamma interaction with a fluorescent SRC-1 peptide, indicating a lack of receptor activation. Therefore, combined approaches using ANS and fluorescent coactivator peptides to monitor PPARgamma binding and interactions may provide valuable strategies to fully understand the role of PPARgamma ligands. Copyright 2009 Elsevier Inc. All rights reserved.

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands.

PubMed

Mogensen, Jesper E; Wimmer, Reinhard; Larsen, Jørgen N; Spangfort, Michael D; Otzen, Daniel E

2002-06-28

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS. Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.

Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study

NASA Astrophysics Data System (ADS)

Das, Sourav; Ghosh, Pooja; Koley, Sudipta; Singha Roy, Atanu

2018-03-01

The interactions of naringenin (NG) and naringin (NR) with Hen Egg White Lysozyme (HEWL) in aqueous medium have been investigated using UV-vis spectroscopy, steady-state fluorescence, circular dichroism (CD), Fourier Transform infrared spectroscopy (FT-IR) and molecular docking analyses. Both NG and NR can quench the intrinsic fluorescence of HEWL via static quenching mechanism. At 300 K, the value of binding constant (Kb) of HEWL-NG complex (5.596 ± 0.063 × 104 M- 1) was found to be greater than that of HEWL-NR complex (3.404 ± 0.407 × 104 M- 1). The negative ΔG° values in cases of both the complexes specify the spontaneous binding. The binding distance between the donor (HEWL) and acceptor (NG/NR) was estimated using the Försters theory and the possibility of non-radiative energy transfer from HEWL to NG/NR was observed. The presence of metal ions (Ca2 +, Cu2 + and Fe2 +) decreased the binding affinity of NG/NR towards HEWL. Synchronous fluorescence studies indicate the change in Trp micro-environment due to the incorporation of NG/NR into HEWL. CD and FT-IR studies indicated that the α-helicity of the HEWL was slightly enhanced due to ligand binding. NG and NR inhibited the enzymatic activity of HEWL and exhibited their affinity for the active site of HEWL. Molecular docking studies revealed that both NG and NR bind in the close vicinity of Trp 62 and Trp 63 residues which is vital for the catalytic activity.

Synthesis and properties of Asante Calcium Red--a novel family of long excitation wavelength calcium indicators.

PubMed

Hyrc, Krzysztof L; Minta, Akwasi; Escamilla, P Rogelio; Chan, Patrick P L; Meshik, Xenia A; Goldberg, Mark P

2013-10-01

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450-540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca(2+)-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (< 490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd = 0.49 ± 0.07 μM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) or Mg2+ (Kd∼5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators. Copyright © 2013 Elsevier Ltd. All rights reserved.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

PubMed

Zhang, Suge; Sun, Hongxia; Chen, Hongbo; Li, Qian; Guan, Aijiao; Wang, Lixia; Shi, Yunhua; Xu, Shujuan; Liu, Meirong; Tang, Yalin

2018-05-01

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells. Copyright © 2018. Published by Elsevier B.V.

Synthesis and Properties of Asante Calcium Red –a Novel Family of Long Excitation Wavelength Calcium Indicators

PubMed Central

Hyrc, Krzysztof L.; Minta, Akwasi; Escamilla, P. Rogelio; Chan, Patrick P.L.; Meshik, Xenia A.; Goldberg, Mark P.

2013-01-01

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (~780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd=0.49±0.07 μM; ACR-1) or low affinity (Kd=6.65±0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd =0.38±0.02 nM) or Mg2+ (Kd ~5 mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=6.31±0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators. PMID:24017967

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

PubMed

Dantas de Araujo, Aline; Wu, Chongyang; Wu, Kai-Chen; Reid, Robert C; Durek, Thomas; Lim, Junxian; Fairlie, David P

2017-06-21

Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (K d ) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pK i of 8.6 ± 0.2 and K i of 2.5 nM) and C3aR ligands TR16 (pK i of 6.8 ± 0.1 and K i of 138 nM), BR103 (pK i of 6.7 ± 0.1 and K i of 185 nM), BR111 (pK i of 6.3 ± 0.2 and K i of 544 nM) and SB290157 (pK i of 6.3 ± 0.1 and K i of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

PubMed

Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

2018-07-05

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

NASA Astrophysics Data System (ADS)

Li, Shihui; Li, Daojin

2011-11-01

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC ( CBZC/ CLys < 9) and a combined quenching process at higher concentration of BZC ( CBZC/ CLys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn 2+ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes.

PubMed

Bottini, Massimo; D'Annibale, Federica; Magrini, Andrea; Cerignoli, Fabio; Arimura, Yutaka; Dawson, Marcia I; Bergamaschi, Enrico; Rosato, Nicola; Bergamaschi, Antonio; Mustelin, Tomas

2007-01-01

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.

A laser scanning confocal imaging-surface plasmon resonance system application in real time detection of antibody-antigen interaction

NASA Astrophysics Data System (ADS)

Zhang, H. Y.; Yang, L. Q.; Liu, W. M.

2011-12-01

The laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, but most LSCM work is qualitative analysis and it is very hard to achieve quantitative detection directly with the changing of the fluorescent intensity. A new real time sensor system for the antibody-antigen interaction detection was built integrating with a LSCM and a wavelength-dependent surface plasmon resonance (SPR) sensor. The system was applied to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibody in real time. The fluorescence images changing is well with that of SPR wavelengths in real time, and the trend of the resonance wavelength shift with the concentrations of antibody is similar to that of the fluorescent intensity changing. The results show that SPR makes up the short of quantificational analysis with LSCM with the high spatial resolution. The sensor system shows the merits of the of the LSCM and SPR synergetic application, which are of great importance for practical application in biosensor and life science for interesting local interaction.

Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

2004-12-01

Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

Measuring the steady-state properties of Ca²⁺ indicators with a set of calibrated [Ca²⁺] solutions.

PubMed

Faas, Guido C; Mody, Istvan

2014-07-01

Fluorescent Ca(2+) indicators are widely used to measure the concentration of free Ca(2+) ([Ca(2+)]free) in biological processes. By calibrating the dye under the same experimental conditions as employed during its planned use, the actual [Ca(2+)] can be calculated from the measured fluorescence. When using non ratiometric dyes, such as the Oregon Green BAPTA (OGB) family of dyes or the Fluo dyes, the steady-state affinity (K(d)) and the ratio between the maximal and minimal fluorescence (F(ratio) = F(max)/F(min)) of the particular dye are needed for this conversion. Although these values are usually given by the manufacturer, we consistently find that the actual values can differ between various batches delivered by the companies that make the dyes. In this protocol, we provide the recipe for a series of solutions with a known and tightly buffered [Ca(2+)](free) and describe how to use these mixtures to determine the exact K(d) and F(ratio) of a fluorescent Ca(2+) dye. © 2014 Cold Spring Harbor Laboratory Press.

Chemical modification of M13 bacteriophage and its application in cancer cell imaging.

PubMed

Li, Kai; Chen, Yi; Li, Siqi; Nguyen, Huong Giang; Niu, Zhongwei; You, Shaojin; Mello, Charlene M; Lu, Xiaobing; Wang, Qian

2010-07-21

The M13 bacteriophage has been demonstrated to be a robust scaffold for bionanomaterial development. In this paper, we report on the chemical modifications of three kinds of reactive groups, i.e., the amino groups of lysine residues or N-terminal, the carboxylic acid groups of aspartic acid or glutamic acid residues, and the phenol group of tyrosine residues, on M13 surface. The reactivity of each group was identified through conjugation with small fluorescent molecules. Furthermore, the regioselectivity of each reaction was investigated by HPLC-MS-MS. By optimizing the reaction condition, hundreds of fluorescent moieties could be attached to create a highly fluorescent M13 bacteriophage. In addition, cancer cell targeting motifs such as folic acid could also be conjugated onto the M13 surface. Therefore, dual-modified M13 particles with folic acid and fluorescent molecules were synthesized via the selective modification of two kinds of reactive groups. Such dual-modified M13 particles showed very good binding affinity to human KB cancer cells, which demonstrated the potential applications of M13 bacteriophage in bioimaging and drug delivery.

Insight into the heterogeneous adsorption of humic acid fluorescent components on multi-walled carbon nanotubes by excitation-emission matrix and parallel factor analysis.

PubMed

Yang, Chenghu; Liu, Yangzhi; Cen, Qiulin; Zhu, Yaxian; Zhang, Yong

2018-02-01

The heterogeneous adsorption behavior of commercial humic acid (HA) on pristine and functionalized multi-walled carbon nanotubes (MWCNTs) was investigated by fluorescence excitation-emission matrix and parallel factor (EEM- PARAFAC) analysis. The kinetics, isotherms, thermodynamics and mechanisms of adsorption of HA fluorescent components onto MWCNTs were the focus of the present study. Three humic-like fluorescent components were distinguished, including one carboxylic-like fluorophore C1 (λ ex /λ em = (250, 310) nm/428nm), and two phenolic-like fluorophores, C2 (λ ex /λ em = (300, 460) nm/552nm) and C3 (λ ex /λ em = (270, 375) nm/520nm). The Lagergren pseudo-second-order model can be used to describe the adsorption kinetics of the HA fluorescent components. In addition, both the Freundlich and Langmuir models can be suitably employed to describe the adsorption of the HA fluorescent components onto MWCNTs with significantly high correlation coefficients (R 2 > 0.94, P< 0.05). The dissimilarity in the adsorption affinity (K d ) and nonlinear adsorption degree from the HA fluorescent components to MWCNTs was clearly observed. The adsorption mechanism suggested that the π-π electron donor-acceptor (EDA) interaction played an important role in the interaction between HA fluorescent components and the three MWCNTs. Furthermore, the values of the thermodynamic parameters, including the Gibbs free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°), showed that the adsorption of the HA fluorescent components on MWCNTs was spontaneous and exothermic. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering single-molecule, nanoscale, and microscale bio-functional materials via click chemistry

NASA Astrophysics Data System (ADS)

Daniele, Michael Angelo-Anthony

To expand the design envelope and supplement the materials library available to biomaterials scientists, the copper(I)-catalyzed azide-alkyne cycloaddition (CuCAAC) was explored as a route to design, synthesize and characterize bio-functional small-molecules, nanoparticles, and microfibers. In each engineered system, the use of click chemistry provided facile, bio-orthogonal control for materials synthesis; moreover, the results provided a methodology and more complete, fundamental understanding of the use of click chemistry as a tool for the synergy of biotechnology, polymer and materials science. Fluorophores with well-defined photophysical characteristics (ranging from UV to NIR fluorescence) were used as building blocks for small-molecule, fluorescent biosensors. Fluorophores were paired to exhibit fluorescence resonant energy transfer (FRET) and used to probe the metabolic activity of carbazole 1,9a-dioxygenase (CARDO). The FRET pair exhibited a significant variation in PL response with exposure to the lysate of Pseudomonas resinovorans CA10, an organism which can degrade variants of both the donor and acceptor fluorophores. Nanoparticle systems were modified via CuCAAC chemistry to carry affinity tags for CARDO and were subsequently utilized for affinity based bioseparation of CARDO from crude cell lysate. The enzymes were baited with an azide-modified carbazolyl-moiety attached to a poly(propargyl acrylate) nanoparticle. Magnetic nanocluster systems were also modified via CuCAAC chemistry to carry fluorescent imaging tags. The iron-oxide nanoclusters were coated with poly(acrylic acid-co-propargyl acrylate) to provide a clickable surface. Ultimately, alternate Cu-free click chemistries were utilized to produce biohybrid microfibers. The biohybrid microfibers were synthesized under benign photopolymerization conditions inside a microchannel, allowing the encapsulation of viable bacteria. By adjusting pre-polymer solutions and laminar flow rates within the microchannel, the morphology, hydration, and thermal properties of the fibers were easily tuned. The methodology produced hydrogel fibers that sustained viable cells as demonstrated by the encapsulation and subsequent proliferation of Bacillus cereus and Escherichia coli communities.

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

NASA Astrophysics Data System (ADS)

Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

2015-11-01

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals. Electronic supplementary information (ESI) available: Schematic representation of PeryBPb1-fused DsRed-Monomer, fluorescence spectra of perylene crystals and DsRed-Monomer, and emission spectra of DsRed-Monomer at various excitation wavelengths. See DOI: 10.1039/c5nr06471f

Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

NASA Astrophysics Data System (ADS)

Gober, Isaiah Nathaniel

This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the possible ways for detecting PTMs and may find use in the development of new assays for enzymes that lack robust methods for measuring their activity. The third section explores the development of new small molecule receptors capable of selectively binding hydrophilic guests in water, such as the lower methylation states of lysine. We identified a receptor, A2I, that has improved binding affinity and selectivity for dimethyllysine (Kme2). The receptor was discovered and synthesized by using dynamic combinatorial chemistry (DCC) to redesign a small molecule receptor (A2B ) that preferentially binds trimethyllysine (Kme3). Incorporating a biphenyl monomer with ortho-di-substituted carboxylates into the receptor lead to the formation of a salt bridge interaction with Kme2. These favorable electrostatic and hydrogen bonding interactions produced a receptor with 32-fold tighter binding to Kme2, which is the highest affinity synthetic receptor for Kme2 in the context of a peptide that has been reported. This work provides insight into effective strategies for binding hydrophilic, cationic guests in water and is an encouraging result toward a synthetic receptor that selectively binds Kme2 over other methylation states of lysine. In the final section, a small molecule receptor for Kme3 (A 2B) was redesigned using DCC to incorporate either aromatic or acidic amino acids into the receptor. We proposed that the incorporation of amino acids could introduce additional non-covalent interactions (such as cation-pi, electrostatic, and hydrogen bonding) with a guest bound inside the pocket of the receptor. However, selective non-covalent interactions between the amino acid side chain on the modified receptor and the bound methylated lysine guest could not be achieved. This is most likely due to the conformational flexibility of the amino acid-functionalized receptors. Furthermore, attaching amino acids to the receptor seemed to increase non-specific electrostatic interactions, resulting in tighter binding to the unmethylated lysine peptide (compared to A2B). Ultimately, this highlights the importance of incorporating monomers with less conformational flexibility that can rigidly place functional groups into the binding pocket.

Aerogel Fingerprint Media

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Fred S.; Andresen, Brian D.

1999-09-21

A fingerprint medium which is made of an aerogel having a predetermined density. The fingerprint medium may have a midrange density for forming plates or may be crushed forming a powder. The fingerprint medium may further include at least one of a metal and metal oxide to enhance characteristics desirable in a fingerprint medium.

A Program Evaluation of a Leadership Academy for School Principals

ERIC Educational Resources Information Center

Wagner, Kristi E.

2014-01-01

This program evaluation focused on mid-range outcomes of a leadership academy for school principals. The mixed-methods evaluation included interviews, principals' instructional observation database, and teacher surveys. The Principal Academy program was designed to build principals' knowledge of high-yield instructional strategies (Hattie, 2009),…

Mission and Assets Database

NASA Technical Reports Server (NTRS)

Baldwin, John; Zendejas, Silvino; Gutheinz, Sandy; Borden, Chester; Wang, Yeou-Fang

2009-01-01

Mission and Assets Database (MADB) Version 1.0 is an SQL database system with a Web user interface to centralize information. The database stores flight project support resource requirements, view periods, antenna information, schedule, and forecast results for use in mid-range and long-term planning of Deep Space Network (DSN) assets.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

PubMed

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

2014-10-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

PubMed Central

Falck, David; de Vlieger, Jon S. B.; Niessen, Wilfried M. A.; Kool, Jeroen; Honing, Maarten; Irth, Hubertus

2010-01-01

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures. Figure P38 α online screening platform Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4087-8) contains supplementary material, which is available to authorized users. PMID:20730527

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Nai-Wei; Gao, Yong; Schill, Megan S.

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirusencoded protein vCCI, a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes, and may represent a potent method to stop inflammation. Previously, our structure of the vCCI:MIP-1β complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1βN-terminus, 20’s region and 40’s loop. However, the interactionsmore » between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), another CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI:MIP-1βcomplex, and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin. Compared to wild-type eotaxin, single, double, or triple mutations at these critical charged residues weaken the binding. One exception is the K47A mutation that exhibits increased affinity for vCCI, which can be explained structurally. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1, MIP-1β and RANTES, were determined as 1.09 nM, 1.16 nM, and 0.22 nM, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and different CC chemokines.« less

Variations in Nuclear Localization Strategies Among Pol X Family Enzymes.

PubMed

Kirby, Thomas W; Pedersen, Lars C; Gabel, Scott A; Gassman, Natalie R; London, Robert E

2018-06-22

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional NLS in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase μ (pol μ), and DNA polymerase λ (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes, and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2011-11-01

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Greatly enhanced binding of a cationic porphyrin towards bovine serum albumin by cucurbit[8]uril.

PubMed

Lei, Wanhua; Jiang, Guoyu; Zhou, Qianxiong; Zhang, Baowen; Wang, Xuesong

2010-10-28

Binding affinity towards serum albumin and intracellular proteins is of importance for a photodynamic therapy (PDT) sensitizer to selectively localize in tumours and efficiently induce cell death. In this paper, it was found that cucurbit[8]uril (CB8) can greatly improve the binding affinity of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), a promising PDT photosensitizer, towards bovine serum albumin (BSA). Absorption, fluorescence emission, (1)H NMR, dynamic light scattering, atomic force microscope, as well as protein photocleavage measurements suggest that the binding enhancement originates from the formation of a ternary complex of CB8·TMPyP·tryptophan residues. This finding opens up a new approach for the development of more efficient PDT agents.

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

PubMed

Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

2007-05-15

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.

PubMed

Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos

2017-05-13

Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

2000-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1998-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry.

PubMed

Hageneder, Simone; Bauch, Martin; Dostalek, Jakub

2016-08-15

This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays - epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 10(2) by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Self-aggregation of bio-surfactants within ionic liquid 1-ethyl-3-methylimidazolium bromide: A comparative study and potential application in antidepressants drug aggregation

NASA Astrophysics Data System (ADS)

Banjare, Manoj Kumar; Behera, Kamalakanta; Kurrey, Ramsingh; Banjare, Ramesh Kumar; Satnami, Manmohan L.; Pandey, Siddharth; Ghosh, Kallol K.

2018-06-01

Aggregation behavior of bio-surfactants (BS) sodium cholate (NaC) and sodium deoxycholate (NaDC) within aqueous solution of ionic liquid (IL) 1-ethyl-3-methylimidazolium bromide [Emim][Br] has been investigated using surface tension, conductivity, steady state fluorescence, FT-IR and dynamic light scattering (DLS) techniques. Various interfacial and thermodynamic parameters are determined in the presence of different wt% of IL [Emim][Br]. Information regarding the local microenvironment and size of the aggregates is obtained from fluorescence and DLS, respectively. FT-IR spectral response is used to reveal the interactions taking place within aqueous NaC/NaDC micellar solutions. It is noteworthy to mention that increasing wt% of [Emim][Br] results in an increase in the spontaneity of micelle formation and the hydrophilic IL shows more affinity for NaC as compared to NaDC. Further, the micellar solutions of BS-[Emim][Br] are utilized for studying the aggregation of antidepressants drug promazine hydrochloride (pH). UV-vis spectroscopic investigation reveals interesting outcomes and the results show changes in spectral absorbance of PH drug on the addition of micellar solution (BS-[Emim][Br]). Highest binding affinity and most promising activity are shown for NaC as compared to NaDC.

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins

NASA Astrophysics Data System (ADS)

Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A.; Jiménez, M. Consuelo

2018-06-01

Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α1-acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222.

Amine-capped ZnS-Mn2+ nanocrystals for fluorescence detection of trace TNT explosive.

PubMed

Tu, Renyong; Liu, Bianhua; Wang, Zhenyang; Gao, Daming; Wang, Feng; Fang, Qunling; Zhang, Zhongping

2008-05-01

Mn2+-doped ZnS nanocrystals with an amine-capping layer have been synthesized and used for the fluorescence detection of ultratrace 2,4,6-trinitrotoluene (TNT) by quenching the strong orange Mn2+ photoluminescence. The organic amine-capped nanocrystals can bind TNT species from solution and atmosphere by the acid-base pairing interaction between electron-rich amino ligands and electron-deficient aromatic rings. The resultant TNT anions bound onto the amino monolayer can efficiently quench the Mn2+ photoluminescence through the electron transfer from the conductive band of ZnS to the lowest unoccupied molecular orbital (LUMO) of TNT anions. The amino ligands provide an amplified response to the binding events of nitroaromatic compounds by the 2- to approximately 5-fold increase in quenching constants. Moreover, a large difference in quenching efficiency was observed for different types of nitroaromatic analytes, dependent on the affinity of nitro analytes to the amino monolayer and their electron-accepting abilities. The amine-capped nanocrystals can sensitively detect down to 1 nM TNT in solution or several parts-per-billion of TNT vapor in atmosphere. The ion-doped nanocrystal sensors reported here show a remarkable air/solution stability, high quantum yield, and strong analyte affinity and, therefore, are well-suited for detecting the ultratrace TNT and distinguishing different nitro compounds.

Inferring Diffusion Dynamics from FCS in Heterogeneous Nuclear Environments

PubMed Central

Tsekouras, Konstantinos; Siegel, Amanda P.; Day, Richard N.; Pressé, Steve

2015-01-01

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show—first using synthetic data—that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell’s nucleus as well as 2) in the cell’s cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins. PMID:26153697

A new fluorescent probe for distinguishing Zn2+ and Cd2+ with high sensitivity and selectivity.

PubMed

Tan, Yiqun; Gao, Junkuo; Yu, Jiancan; Wang, Ziqi; Cui, Yuanjing; Yang, Yu; Qian, Guodong

2013-08-28

A new fluorescence probe for distinguishing Zn(2+) and Cd(2+) is designed and synthesized. For the first time to our knowledge, this probe can recognize similar metal ions by coherently utilizing intramolecular charge transfer (ICT) and different electronic affinities of various metal ions, instead of by selective coordination alone, which may be interfered with and lose its selectivity easily in a complicated environment, providing a distinct recognition even by the naked eye for Zn(2+) and Cd(2+) with the sensitivity at the ppb level. This design strategy may initiate a straightforward approach for the selective detection of various metal ions with similar chemical properties in extensive applications such as environmental, industrial, and bio-science.

Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA.

PubMed

Nozeret, Karine; Bonan, Marc; Yarmoluk, Serguiy M; Novopashina, Darya S; Boutorine, Alexandre S

2015-09-01

Synthetic minor groove-binding pyrrole-imidazole polyamides labeled by fluorophores are promising candidates for fluorescence imaging of double-stranded DNA in isolated chromosomes or fixed and living cells. We synthesized nine hairpin and two head-to-head tandem polyamides targeting repeated sequences from mouse major satellites. Their interaction with synthetic target dsDNA has been studied by physico-chemical methods in vitro before and after coupling to various fluorophores. Great variability in affinities and fluorescence properties reveals a conclusion that these properties do not only rely on recognition rules, but also on other known and unknown structural factors. Individual testing of each probe is needed before cellular applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to hydrophilic microspheres: development of a mobile microscale UV light actinometer.

PubMed

Fang, Shiyue; Guan, Yousheng; Blatchley, Ernest R; Shen, Chengyue; Bergstrom, Donald E

2008-03-01

( E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine was biotinylated through a diisopropylsilylacetal linkage and attached to the surface of hydrophilic streptavidin-coated microspheres through the high-affinity noncovalent interaction between biotin and streptavidin. The functionalized microspheres form a stable suspension in water. Upon UV irradiation, the nonfluorescent ( E)-5-[2-(methoxycarbonyl)ethenyl]cytidine on the microspheres undergoes photocyclization to produce highly fluorescent 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine. The fluorescence intensity of the microspheres can be correlated to the particle-specific UV doses applied at different suspension concentrations. The microspheres allow one to measure the UV dose (fluence) distribution in high-throughput water disinfection systems.

Evaluation of a metalloporphyrin (THPPMnCl) for necrosis-affinity in rat models of necrosis.

PubMed

Li, Yue; Liu, Xuejiao; Zhang, Dongjian; Lou, Bin; Peng, Fei; Wang, Xiaoning; Shan, Xin; Jiang, Cuihua; Gao, Meng; Sun, Ziping; Ni, Yicheng; Huang, Dejian; Zhang, Jian

2015-12-01

The combination of an (13I)I-labeled necrosis-targeting agent (NTA) with a vascular disrupting agent is a novel and potentially powerful technique for tumor necrosis treatment (TNT). The purpose of this study was to evaluate a NTA candidate, THPPMnCl, using (131)I isotope for tracing its biodistribution and necrosis affinity. (131)I-THPPMnCl was intravenously injected in rat models with liver, muscle, and tumor necrosis and myocardial infarction (MI), followed by investigations with macroscopic autoradiography, triphenyltetrazolium chloride (TTC) histochemical staining, fluorescence microscopy and H&E stained histology for up to 9 days. (131)I-THPPMnCl displayed a long-term affinity for all types of necrosis and accumulation in the mononuclear phagocytic system especially in the liver. Autoradiograms and TTC staining showed a good targetability of (131)I-THPPMnCl for MI. These findings indicate the potential of THPPMnCl for non-invasive imaging assessment of necrosis, such as in MI. However, (13I)I-THPPMnCl is unlikely suitable for TNT due to its long-term retention in normal tissues.

Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

NASA Astrophysics Data System (ADS)

Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2006-07-01

Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

2-Phenylbenzothiazole conjugated with cyclopentadienyl tricarbonyl [CpM(CO)3] (M = Re, (99m)Tc) complexes as potential imaging probes for β-amyloid plaques.

PubMed

Jia, Jianhua; Cui, Mengchao; Dai, Jiapei; Liu, Boli

2015-04-14

Technetium-99m-labeled cyclopentadienyl tricarbonyl complexes conjugated with the 2-phenylbenzothiazole binding motif were synthesized. The rhenium surrogates , , and were demonstrated to have moderate to high affinities for Aβ1-42 aggregates with Ki values of 142, 76, 64 and 24 nM, respectively. During the fluorescence staining of brain sections of transgenic mice and patients with Alzheimer's disease, these rhenium complexes demonstrated perfect and intense labeling of Aβ plaques. Moreover, in in vitro autoradiography, (99m)Tc-labeled complexes clearly detected β-amyloid plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these tracers for Aβ plaques. However, these compounds did not show desirable properties in vivo, especially showing poor brain uptake (below 0.5% ID g(-1)), which will hinder the further development of these tracers as brain imaging agents. Nonetheless, it is encouraging that these (99m)Tc-labeled complexes designed by a conjugate approach displayed sufficient affinities for Aβ plaques.

Comparative analysis the binding affinity of mycophenolic sodium and meprednisone with human serum albumin: Insight by NMR relaxation data and docking simulation.

PubMed

Ma, Xiaoli; He, Jiawei; Yan, Jin; Wang, Qing; Li, Hui

2016-03-25

Mycophenolic sodium is an immunosuppressive agent that is always combined administration with corticosteroid in clinical practice. Considering the distribution and side-effect of the drug may change when co-administrated drug exist, this paper comparatively analyzed the binding ability of mycophenolic sodium and meprednisone toward human serum albumin by nuclear magnetic resonance relaxation data and docking simulation. The nuclear magnetic resonance approach was based on the analysis of proton selective and non-selective relaxation rate enhancement of the ligand in the absence and presence of macromolecules. The contribution of the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index. This approach allowed the comparison of the binding affinity of mycophenolic sodium and meprednisone. Molecular modeling was operated to simulate the binding model of ligand and albumin through Autodock 4.2.5. Competitive binding of mycophenolic sodium and meprednisone was further conducted through fluorescence spectroscopy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

PubMed

Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

2014-07-03

Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+ Antiport 1

PubMed Central

Barkla, Bronwyn J.; Charuk, Jeffrey H. M.; Cragoe, Edward J.; Blumwald, Eduardo

1990-01-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. Images Figure 7 PMID:16667602

Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

PubMed

Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

1990-07-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

Arrestin binds to different phosphorylated regions of the thyrotropin-releasing hormone receptor with distinct functional consequences.

PubMed

Jones, Brian W; Hinkle, Patricia M

2008-07-01

Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

PubMed Central

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

A carbon dot-based "off-on" fluorescent probe for highly selective and sensitive detection of phytic acid.

PubMed

Gao, Zhao; Wang, Libing; Su, Rongxin; Huang, Renliang; Qi, Wei; He, Zhimin

2015-08-15

We herein report a facile, one-step pyrolysis synthesis of photoluminescent carbon dots (CDs) using citric acid as the carbon source and lysine as the surface passivation reagent. The as-prepared CDs show narrow size distribution, excellent blue fluorescence and good photo-stability and water dispersivity. The fluorescence of the CDs was found to be effectively quenched by ferric (Fe(III)) ions with high selectivity via a photo-induced electron transfer (PET) process. Upon addition of phytic acid (PA) to the CDs/Fe(III) complex dispersion, the fluorescence of the CDs was significantly recovered, arising from the release of Fe(III) ions from the CDs/Fe(III) complex because PA has a higher affinity for Fe(III) ions compared to CDs. Furthermore, we developed an "off-on" fluorescence assay method for the detection of phytic acid using CDs/Fe(III) as a fluorescent probe. This probe enables the selective detection of PA with a linear range of 0.68-18.69 μM and a limit of detection (signal-to-noise ratio is 3) of 0.36 μM. The assay method demonstrates high selectivity, repeatability, stability and recovery ratio in the detection of the standard and real PA samples. We believe that the facile operation, low-cost, high sensitivity and selectivity render this CD-based "off-on" fluorescent probe an ideal sensing platform for the detection of PA. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of fluorescent acridine dyes acridine orange and 9-aminoacridine to hemoglobin: Elucidation of their molecular recognition by spectroscopy, calorimetry and molecular modeling techniques.

PubMed

Chatterjee, Sabyasachi; Kumar, Gopinatha Suresh

2016-06-01

The molecular interaction between hemoglobin (HHb), the major human heme protein, and the acridine dyes acridine orange (AO) and 9-aminoacridine (9AA) was studied by various spectroscopic, calorimetric and molecular modeling techniques. The dyes formed stable ground state complex with HHb as revealed from spectroscopic data. Temperature dependent fluorescence data showed the strength of the dye-protein complexation to be inversely proportional to temperature and the fluorescence quenching was static in nature. The binding-induced conformational change in the protein was investigated using circular dichroism, synchronous fluorescence, 3D fluorescence and FTIR spectroscopy results. Circular dichroism data also quantified the α-helicity change in hemoglobin due to the binding of acridine dyes. Calorimetric studies revealed the binding to be endothermic in nature for both AO and 9AA, though the latter had higher affinity, and this was also observed from spectroscopic data. The binding of both dyes was entropy driven. pH dependent fluorescence studies revealed the existence of electrostatic interaction between the protein and dye molecules. Molecular modeling studies specified the binding site and the non-covalent interactions involved in the association. Overall, the results revealed that a small change in the acridine chromophore leads to remarkable alteration in the structural and thermodynamic aspects of binding to HHb. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of tumor-targeted near infrared probes for fluorescence guided surgery.

PubMed

Kelderhouse, Lindsay E; Chelvam, Venkatesh; Wayua, Charity; Mahalingam, Sakkarapalayam; Poh, Scott; Kularatne, Sumith A; Low, Philip S

2013-06-19

Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance.

PubMed

Li, Shihui; Li, Daojin

2011-11-01

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C(BZC)/C(Lys)<9) and a combined quenching process at higher concentration of BZC (C(BZC)/C(Lys)>9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn(2+) on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1.

PubMed

Tugaeva, Kristina V; Faletrov, Yaroslav V; Allakhverdiev, Elvin S; Shkumatov, Vladimir M; Maksimov, Eugene G; Sluchanko, Nikolai N

2018-02-26

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

A Comparative Analysis of the Budget Process in the Venezuelan and U.S. Navies.

DTIC Science & Technology

1979-12-01

accounting problems in particular agencies. The DINCA developed a document, Sistema de Contabilidad de la Ejecucion Financiera del Presupuesto para...orienta- tion of the "Plan Operativo Anual" (POA) - Operative Annual Plan. During the Navy’s mid-range planning process the POA is produced. By means of

Application of Psychometric Theory to the Measurement of Voice Quality Using Rating Scales

ERIC Educational Resources Information Center

Shrivastav, Rahul; Sapienza, Christine M.; Nandur, Vuday

2005-01-01

Rating scales are commonly used to study voice quality. However, recent research has demonstrated that perceptual measures of voice quality obtained using rating scales suffer from poor interjudge agreement and reliability, especially in the midrange of the scale. These findings, along with those obtained using multidimensional scaling (MDS), have…

Muscle Dysmorphia among College Men: An Emerging Gender-Related Counseling Concern

ERIC Educational Resources Information Center

Davey, Carla M.; Bishop, John B.

2006-01-01

Recent literature suggests that, like midrange eating disorders among college women, male muscle dysmorphia is emerging as a physical as well as a health concern among college men. The authors define the disorder, review diagnostic and etiological considerations, and discuss the added complication of creatine use to self-manage muscle dysmorphic…

Application of 2D Correlation Spectroscopy with MCR in the Preparation of Glycerol Polyesters

USDA-ARS?s Scientific Manuscript database

The condensation of glycerol and adipic acid was studied by midrange FTIR to identify spectral changes associated with the polymerization reaction. This biobased polymer is being evaluated for use as a controlled release matrix where the extent of reaction is a key performance parameter. A spectrosc...

Learning Hebrew by Writing in English

ERIC Educational Resources Information Center

Jacobson, Rolf A.

2011-01-01

This essay explores a midrange teaching and learning issue regarding the teaching of biblical languages and one strategy for addressing the issue. Seminary students do not yield a great enough return in exchange for the investment they are required to make in learning biblical languages. Students invest great time and money, but they do not learn…

Tipping Points? Curvilinear Associations between Activity Level and Mental Development in Toddlers

ERIC Educational Resources Information Center

Flom, Megan; Cohen, Madeleine; Saudino, Kimberly J.

2017-01-01

The Theory of Optimal Stimulation (Zentall & Zentall, "Psychological Bulletin," 94, 1983, 446) posits that the relation between activity level (AL) and cognitive performance follows an inverted U shape where midrange AL predicts better cognitive performance than AL at the extremes. We explored this by fitting linear and quadratic…

An Exploratory Study of the Relationships between Family Functioning and Parenting Styles: The Perceptions of Mothers of Young Grade School Children.

ERIC Educational Resources Information Center

Mupinga, Emily Evellyne; Garrison, M. E. Betsy; Pierce, Sarah H.

2002-01-01

A study of 151 mothers of elementary students identified relationships between parenting styles (authoritative, authoritarian, permissive) and family functioning (adaptability, cohesion). Families with balanced and moderately balanced levels of adaptability and cohesion had higher levels of authoritative parenting. Midrange balance was associated…

Study on the interaction of a cyanine dye with human serum transferrin.

PubMed

Zhang, Xiu-feng; Chen, Lei; Yang, Qian-fan; Li, Qian; Sun, Xiao-ran; Chen, Hong-bo; Yang, Guang; Tang, Ya-lin

2015-12-01

Complexation between the primary carrier of ligands in blood plasma, human serum transferrin (Tf), and a cyanine dye, 3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-phenyl-thiacarbocyanine-triethylam monium salt (PTC) was investigated using fluorescence spectra, UV/Vis absorption spectra, synchronous fluorescence spectra, circular dichroism (CD) and molecular dynamic docking. The experimental results demonstrate that the formation of PTC-Tf complex is stabilized by van der Waal's interactions and hydrogen bonds, and the binding constants were found to be 8.55 × 10(6), 8.19 × 10(6) and 1.75 × 10(4) M(-1). Moreover, fluorescence experiments prove that the operational mechanism for the fluorescence quenching is static quenching and non-radiative energy transfer. Structural investigation of the PTC-Tf complexes via synchronous fluorescence spectra and CD showed that the structure of Tf became more stable with a major increase in the α-helix content and increased polarity around the tryptophan residues after PTC binding. In addition, molecular modeling highlights the residues located in the N-lobe, which retain high affinity for PTC. The mode of action of the PTC-Tf complex is illustrated by these results, and may provide an effective pathway for the transport and targeted delivery of antitumor agents. Copyright © 2015 John Wiley & Sons, Ltd.

Turn-on theranostic fluorescent nanoprobe by electrostatic self-assembly of carbon dots with doxorubicin for targeted cancer cell imaging, in vivo hyaluronidase analysis, and targeted drug delivery.

PubMed

Gao, Na; Yang, Wen; Nie, Hailiang; Gong, Yunqian; Jing, Jing; Gao, Loujun; Zhang, Xiaoling

2017-10-15

This paper reports a turn-on theranostic fluorescent nanoprobe P-CDs/HA-Dox obtained by electrostatic assembly of polyethylenimine (PEI)-modified carbon dots (P-CDs) and Hyaluronic acid (HA)-conjugated doxorubicin (Dox) for hyaluronidase (HAase) detection, self-targeted imaging and drug delivery. P-CDs/HA-Dox show weak emission in a physiological environment. By utilizing the high affinity of HA to CD44 receptors overexpressed on many cancer cells, P-CDs/HA-Dox are capable of targeting and penetrating into cancer cells, where they are activated by HAase. As a result, HA-Dox can be digested into small fragments, causing the release of Dox and thereby restoring the fluorescence of P-CDs. The theranostic fluorescent nanoprobe can effectively distinguish cancer cells from normal cells. The as-prepared nanoprobe achieves a sensitive assay of HAase with a detection limit of 0.65UmL -1 . Furthermore, upon Dox release, the Dox could efficiently induce apoptosis in HeLa cells, as confirmed by MTT assay. The design of such a turn-on theranostic fluorescent probe provides a new strategy for self-targeted and image-guided chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Selective and Sensitive Detection of Cyanide Based on the Displacement Strategy Using a Water-Soluble Fluorescent Probe.

PubMed

La, Ming; Hao, Yuanqiang; Wang, Zhaoyang; Han, Guo-Cheng; Qu, Lingbo

2016-01-01

A water-soluble fluorescent probe (C-GGH) was used for the highly sensitive and selective detection of cyanide (CN(-)) in aqueous media based on the displacement strategy. Due to the presence of the recognition unit GGH (Gly-Gly-His), the probe C-GGH can coordinate with Cu(2+) and consequently display ON-OFF type fluorescence response. Furthermore, the in situ formed nonfluorescent C-GGH-Cu(2+) complex can act as an effective OFF-ON type fluorescent probe for sensing CN(-) anion. Due to the strong binding affinity of CN(-) to Cu(2+), CN(-) can extract Cu(2+) from C-GGH-Cu(2+) complex, leading to the release of C-GGH and the recovery of fluorescent emission of the system. The probe C-GGH-Cu(2+) allowed detection of CN(-) in aqueous solution with a LOD (limit of detection) of 0.017 μmol/L which is much lower than the maximum contaminant level (1.9 μmol/L) for CN(-) in drinking water set by the WHO (World Health Organization). The probe also displayed excellent specificity for CN(-) towards other anions, including F(-), Cl(-), Br(-), I(-), SCN(-), PO4 (3-), N3 (-), NO3 (-), AcO(-), SO4 (2-), and CO3 (2-).

Selective and Sensitive Detection of Cyanide Based on the Displacement Strategy Using a Water-Soluble Fluorescent Probe

PubMed Central

La, Ming; Hao, Yuanqiang; Wang, Zhaoyang; Han, Guo-Cheng; Qu, Lingbo

2016-01-01

A water-soluble fluorescent probe (C-GGH) was used for the highly sensitive and selective detection of cyanide (CN−) in aqueous media based on the displacement strategy. Due to the presence of the recognition unit GGH (Gly-Gly-His), the probe C-GGH can coordinate with Cu2+ and consequently display ON-OFF type fluorescence response. Furthermore, the in situ formed nonfluorescent C-GGH-Cu2+ complex can act as an effective OFF-ON type fluorescent probe for sensing CN− anion. Due to the strong binding affinity of CN− to Cu2+, CN− can extract Cu2+ from C-GGH-Cu2+ complex, leading to the release of C-GGH and the recovery of fluorescent emission of the system. The probe C-GGH-Cu2+ allowed detection of CN− in aqueous solution with a LOD (limit of detection) of 0.017 μmol/L which is much lower than the maximum contaminant level (1.9 μmol/L) for CN− in drinking water set by the WHO (World Health Organization). The probe also displayed excellent specificity for CN− towards other anions, including F−, Cl−, Br−, I−, SCN−, PO4 3−, N3 −, NO3 −, AcO−, SO4 2−, and CO3 2−. PMID:26881185

Reversible and Dynamic Fluorescence Imaging of Cellular Redox Self-Regulation Using Fast-Responsive Near-Infrared Ge-Pyronines.

PubMed

Nie, Hailiang; Jing, Jing; Tian, Yong; Yang, Wen; Zhang, Rubo; Zhang, Xiaoling

2016-04-13

Cellular self-regulation of reactive oxygen species (ROS) stress via glutathione (GSH) antioxidant repair plays a crucial role in maintaining redox balance, which affects various physiological and pathological pathways. In this work, we developed a simple yet effective strategy for reversible, dynamic, and real-time fluorescence imaging of ROS stress and GSH repair, based on novel Ge-pyronine dyes (GePs). Unlike the current O-pyronine (OP) dye, the fluorescence of GePs can be quenched in GSH reduction and then greatly restored by ROS (e.g., ClO(-), ONOO(-), and HO(•)) oxidation because of their unique affinity toward thiols. The "on-off" and "off-on" fluorescence switch can complete in 10 and 20 s, respectively, and exhibit excellent reversibility in vitro and in cells. GePs also show excitation in the long wavelength from the deep-red to near-infrared (NIR) (621-662 nm) region, high fluorescence quantum yield (Φ(fl) = 0.32-0.44) in aqueous media, and excellent cell permeability. Our results demonstrated that GePs can be used for real-time monitoring of the reversible and dynamic interconversion between ROS oxidation and GSH reduction in living cells. GePs might be a useful tool for investigating various redox-related physiological and pathological pathways.

Detection of adenosine triphosphate in HeLa cell using capillary electrophoresis-laser induced fluorescence detection based on aptamer and graphene oxide.

PubMed

Fang, Bi-Yun; Yao, Ming-Hao; Wang, Chun-Yuan; Wang, Chao-Yang; Zhao, Yuan-Di; Chen, Fang

2016-04-01

A method for ATP quantification based on dye-labeled aptamer/graphene oxide (aptamer/GO) using capillary electrophoresis-laser induced fluorescence (CE-LIF) detecting technique has been established. In this method, the carboxyfluorescein (FAM)-labelled ATP aptamers were adsorbed onto the surface of GO, leading to the fluorescence quenching of FAM; after the incubation with a limited amount of ATP, stronger affinity between ATP aptamer and ATP resulted in the desorption of aptamers and the fluorescence restoration of FAM. Then, aptamer-ATP complex and excess of aptamer/GO and GO were separated and quantified by CE-LIF detection. It was shown that a linear relation was existing in the CE-LIF peak intensity of aptamer-ATP and ATP concentration in range of 10-700 μM, the regression equation was F=1.50+0.0470C(ATP) (R(2)=0.990), and the limit of detection was 1.28 μM (3S/N, n=5), which was one order magnitude lower than that of detection in solution by fluorescence method. The approach with excellent specificity and reproducibility has been successfully applied to detecting concentration of ATP in HeLa cell. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced binding of hydrophobic organic contaminants by microwave-assisted humification of soil organic matter.

PubMed

Hur, Jin; Park, Sung-Won; Kim, Min Chan; Kim, Han S

2013-11-01

Enhanced binding of hydrophobic organic contaminants (HOCs) with soil organic matter (SOM) by microwave (MW) irradiation was investigated in this study. We used fluorescence excitation emission matrix, humification index (HIX), and organic carbon partitioning coefficient (Koc) to examine characteristic changes in SOM and its sorptive capacity for HOCs. When MW was irradiated to soils, protein-like fluorescence decreased but fulvic- and humic-like fluorescence increased. The addition of activated carbon in the presence of oxygen facilitated the humification-like alteration of SOM more significantly, evidenced by increases in fulvic- and humic-like fluorescence signals. The extent of SOM-phenanthrene binding also increased with MW treatment, supported by a notable increase in Koc value from 1.8×10(4) to 7.3×10(5)Lkg(-1). Various descriptors indicating the physical and chemical properties of SOM along with the relative percentage of humic-like fluorescence and HIX values demonstrated strong linear relationships with Koc values. These linear relationships indicated that the increased binding affinity of SOM for phenanthrene was attributed to enhanced SOM humification, which was stimulated by MW irradiation. Thus, our results demonstrate that MW irradiation could be effectively used for remediation or for assessing the environmental risks of HOC-contaminated soils and groundwater. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ultrasensitive detection of amifostine and alkaline phosphatase based on the growth of CdS quantum dots.

PubMed

Na, Weidan; Liu, Siyu; Liu, Xiaotong; Su, Xingguang

2015-11-01

In this study, we reported a simple and sensitive fluorescence nanosensor for rapid detection of amifostine and alkaline phosphatase (ALP). The novel nanosensor was based on the fluorescence "turn on-off" of CdS quantum dots (QDs). Firstly, Cd(2+) cation could react with S(2-) anion to generate fluorescent CdS QDs in the presence of amifostine. The fluorescence (FL) intensity of amifostine-capped CdS QDs (Amifostine-CdS QDs) was increased with the increasing amounts of amifostine, and could be used for amifostine detection. However, amifostine could be converted to 2-(3-aminopropylamino) ethanethiol (WR1065) in the presence of ALP based on the dephosphorylation of ALP. Under the optimum conditions, the affinity of WR1065 to CdS QDs was weaker than that of amifostine. Therefore the new generation of WR1065-CdS QDs would reduce the FL intensity with the increase of ALP concentration, and the fluorescence of CdS QDs was turn off. The metabolic process of amifostine in the presence of alkaline phosphatase could be also studied via the change of FL intensity of CdS QDs. The present method was cost-effective, convenient, and does not require any complicated synthetic procedures. Copyright © 2015 Elsevier B.V. All rights reserved.

A Metal-Polydopamine Framework (MPDA) as an Effective Fluorescent Quencher for Highly Sensitive Detection of Hg (II) And Ag (I) ions Through Exonuclease III Activity.

PubMed

Ravikumar, Ayyanu; Panneerselvam, Perumal; Morad, Norhashimah

2018-05-24

In this paper, we propose a metal-polydopamine framework (MPDA) with specific molecular probe which appears to be the most promising approach to a strong fluorescence quencher. The MPDA framework quenching ability towards various organic fluorophore such as aminoethylcomarin acetate (AMCA), 6-carboxyfluorescein (FAM), carboxyteramethylrhodamine (TAMRA) and Cy5 are used to establish a fluorescent biosensor that can selectively recognize Hg2+ and Ag+ ion. The fluorescent quenching efficiency was sufficient to achieve more than 96%. The MPDA framework also exhibits different affinities with ssDNA and dsDNA. In addition, the FAM labelled ssDNA was adsorbed onto MPDA framework, based on their interaction with the complex formed between MPDA frameworks/ssDNA taken as a sensing platform. By taking advantage of this sensor highly sensitive and selective determination of Hg2+and Ag+ ions is achieved through Exonuclease III signal amplification activity. The detection limits of Hg2+and Ag+ achieved to be 1.2 pM and 34 pM respectively, were compared to co-existing metal ions and GO based sensors. Furthermore, the potential applications of this study establish the highly sensitive fluorescence detection targets in environmental and biological fields.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

PubMed Central

Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2011-01-01

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C.

PubMed

Badr, Myriam A; Pinto, Jose R; Davidson, Michael W; Chase, P Bryant

2016-01-01

Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus) was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

Synthesis, photophysical and cytotoxicity evaluations of DNA targeting agents based on 3-amino-1,8-naphthalimide derived Tröger's bases.

PubMed

Murphy, Samantha; Bright, Sandra A; Poynton, Fergus E; McCabe, Thomas; Kitchen, Jonathan A; Veale, Emma B; Williams, D Clive; Gunnlaugsson, Thorfinnur

2014-09-14

The synthesis and characterisation of five bis-1,8-naphthalimide containing Tröger's bases 1–5 formed from their corresponding 3-amino-1,8-naphthalimide precursors 6–10 is described. The photophysical investigations of 1–5 and 6–10 were carried out in several organic solvents as well as in water and as a function of pH using UV-Vis absorption and fluorescence spectroscopies. The DNA binding affinities of 1–5 in aqueous solution at pH 7.4 were also investigated using several UV-Vis absorption and fluorescence experiments by using calf thymus DNA (ct-DNA). These molecules exhibited significant DNA binding affinities; where large binding values (Kb) in the range of 10(6) M(−1) were determined, even in competitive media (50 mM and 160 mM NaCl at pH 7.4). Thermal denaturation measurements also showed that 1–5 significantly stabilised the DNA helix. Using linear and circular dichroism we further demonstrated that the DNA binding interaction occurs both by intercalation and by groove binding. The Tröger's bases were further shown to be rapidly taken up into cells using confocal fluorescence spectroscopy; and cytotoxic studies in HeLa and MCF-7 cells showed that most of the Tröger's bases were effective cytotoxic agents with EC50 values of between 1.1–12 μM and that all the active compounds induced programmed cell death by apoptosis, where up to 70% cellular death was observed after 24 h of incubation for 4.

The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.

PubMed

Oakley, Robert H; Hudson, Christine C; Cruickshank, Rachael D; Meyers, Diane M; Payne, Richard E; Rhem, Shay M; Loomis, Carson R

2002-11-01

G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V(2) receptor (V(2)R), which binds arrestin with high affinity, and the beta(2)-adrenergic receptor (beta(2)AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V(2)R or the beta(2)AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V(2)R and beta(2)AR, respectively. EC(50) values agreed closely with K(d) values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC(50) doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z' values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.

Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry

PubMed Central

Brauchle, Michael; Hansen, Simon; Caussinus, Emmanuel; Lenard, Anna; Ochoa-Espinosa, Amanda; Scholz, Oliver; Sprecher, Simon G.; Plückthun, Andreas; Affolter, Markus

2014-01-01

ABSTRACT Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level. PMID:25416061

Estrogenicity of halogenated bisphenol A: in vitro and in silico investigations.

PubMed

Zhang, Jie; Li, Tiezhu; Wang, Tuoyi; Yuan, Cuiping; Zhong, Shuning; Guan, Tianzhu; Li, Zhuolin; Wang, Yongzhi; Yu, Hansong; Luo, Quan; Wang, Yongjun; Zhang, Tiehua

2018-03-01

The binding interactions of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to human estrogen receptor α ligand binding domain (hERα-LBD) was investigated using a combined in vitro and in silico approach. First, the recombinant hERα-LBD was prepared as a soluble protein in Escherichia coli BL21(DE3)pLysS. A native fluorescent phytoestrogen, coumestrol, was employed as tracer for the fluorescence polarization assay. The results of the in vitro binding assay showed that bisphenol compounds could bind to hERα-LBD as the affinity ligands. All the tested halogenated BPAs exhibited weaker receptor binding than BPA, which might be explained by the steric effect of substituents. Molecular docking studies elucidated that the halogenated BPAs adopted different conformations in the flexible hydrophobic ligand binding pocket (LBP), which is mainly dependent on their distinct halogenation patterns. The compounds with halogen substituents on the phenolic rings and on the bridging alkyl moiety acted as agonists and antagonists for hERα, respectively. Interestingly, all the compounds in the agonist conformation of hERα formed a hydrogen bond with His524, while the compounds in the antagonist conformation formed a hydrogen bond with Thr347. These docking results suggested a pivotal role of His524/Thr347 in maintaining the hERα structure in the biologically active agonist/antagonist conformation. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation, which might be applicable for the structure-based design of novel bisphenol compounds with reduced toxicities and for environmental risk assessment. In addition, based on hERα-LBD as a recognition element, the proposed fluorescence polarization assay may offer an alternative to chromatographic techniques for the multi-residue determination of bisphenol compounds.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, A.N.; Benson, S.C.

1998-07-21

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye. 10 figs.

Modular Bioconjugates to Study Herceptin Resistance: A Structural and Functional Approach

DTIC Science & Technology

2015-10-01

MS2 capsid (Figure 3a on page 1593 of Appendix A). Furthermore, a Kd was determined for our construct that indicated that these constructs maintain...their binding affinity upon attachment to the capsid (Figure 3b,c on page 1593 of Appendix A). Furthermore, live-cell confocal microscopy clearly...1590−1596 1593 parameters to be measured simultaneously.45 This number represents a dramatic increase over traditional fluorescence- based flow

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PubMed

Walkup, Ward G; Kennedy, Mary B

2014-06-01

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

PubMed Central

Senning, Eric N.; Aman, Teresa K.

2016-01-01

Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane. PMID:26755772

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

Hybrid Integrated Silicon Microfluidic Platform for Fluorescence Based Biodetection.

PubMed

Chandrasekaran, Arvind; Acharya, Ashwin; You, Jian Liang; Soo, Kim Young; Packirisamy, Muthukumaran; Stiharu, Ion; Darveau, André

2007-09-11

The desideratum to develop a fully integrated Lab-on-a-chip device capable ofrapid specimen detection for high throughput in-situ biomedical diagnoses and Point-of-Care testing applications has called for the integration of some of the novel technologiessuch as the microfluidics, microphotonics, immunoproteomics and Micro ElectroMechanical Systems (MEMS). In the present work, a silicon based microfluidic device hasbeen developed for carrying out fluorescence based immunoassay. By hybrid attachment ofthe microfluidic device with a Spectrometer-on-chip, the feasibility of synthesizing anintegrated Lab-on-a-chip type device for fluorescence based biosensing has beendemonstrated. Biodetection using the microfluidic device has been carried out usingantigen sheep IgG and Alexafluor-647 tagged antibody particles and the experimentalresults prove that silicon is a compatible material for the present application given thevarious advantages it offers such as cost-effectiveness, ease of bulk microfabrication,superior surface affinity to biomolecules, ease of disposability of the device etc., and is thussuitable for fabricating Lab-on-a-chip type devices.

A light-up probe targeting for Bcl-2 2345 G-quadruplex DNA with carbazole TO

NASA Astrophysics Data System (ADS)

Gu, Yingchun; Lin, Dayong; Tang, Yalin; Fei, Xuening; Wang, Cuihong; Zhang, Baolian; Zhou, Jianguo

2018-02-01

As its significant role, the selective recognition of G-quadruplex with specific structures and functions is important in biological and medicinal chemistry. Carbazole derivatives have been reported as a kind of fluorescent probe with many excellent optical properties. In the present study, the fluorescence of the dye (carbazole TO) increased almost 70 fold in the presence of bcl-2 2345 G4 compared to that alone in aqueous buffer condition with almost no fluorescence and 10-30 fold than those in the presence of other DNAs. The binding study results by activity inhibition of G4/Hemin peroxidase experiment, NMR titration and molecular docking simulation showed the high affinity and selectivity to bcl-2 2345 G4 arises from its end-stacking interaction with G-quartet. It is said that a facile approach with excellent sensitive, good selectivity and quick response for bcl-2 2345 G-quadruplex was developed and may be used for antitumor recognition or antitumor agents.

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain.

PubMed Central

Moores, J C; Magazin, M; Ditta, G S; Leong, J

1984-01-01

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin. Images PMID:6690426

Supercritical angle fluorescence as a tool to study the interaction between lipid bilayer and peptides

NASA Astrophysics Data System (ADS)

Dubois, Valentin; Serrano, Diana; Seeger, Stefan

2017-06-01

The understanding of processes occurring at the interface between two media are of prior importance in various fields of research, from material sciences to biology. A custom-made microscope objective based on the supercritical angle technique was developed in our group, allowing to probe these interfacial events by carrying out surface-sensitive and low invasive spectroscopy of aqueous samples. A biological example of particular interest is the comprehension of neurodegenerative diseases which seem caused by the interaction of specific peptides with the membrane of the neurons. Taking advantage of our optical setup, we used supercritical angle fluorescence spectroscopy to specifically monitor the interaction between a supported lipid bilayer (SLB) and the Amyloid β peptide, notably responsible of the Alzheimer disease. Different forms of the peptide (40 and 42 amino acids composition) were tested and the interfacial fluorescence measured to get information about the lipid integrity and mobility. The adsorption of the peptide was also characterized in terms of kinetic and affinity.

Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

PubMed Central

Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

2014-01-01

Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

Steroids in house sparrows (Passer domesticus): Effects of POPs and male quality signalling.

PubMed

Nossen, Ida; Ciesielski, Tomasz M; Dimmen, Malene V; Jensen, Henrik; Ringsby, Thor Harald; Polder, Anuschka; Rønning, Bernt; Jenssen, Bjørn M; Styrishave, Bjarne

2016-03-15

At high trophic levels, environmental contaminants have been found to affect endocrinological processes. Less attention has been paid to species at lower trophic levels. The house sparrow (Passer domesticus) may be a useful model for investigating effects of POPs in mid-range trophic level species. In male house sparrows, ornamental traits involved in male quality signalling are important for female selection. These traits are governed by endocrinological systems, and POPs may therefore interfere with male quality signalling. The aim of the present study was to use the house sparrow as a mid-range trophic level model species to study the effects of environmental contaminants on endocrinology and male quality signalling. We analysed the levels of selected PCBs, PBDEs and OCPs and investigated the possible effects of these contaminants on circulating levels of steroid hormones (4 progestagens, 4 androgens and 3 estrogens) in male and female adult house sparrows from a population on the island Leka, Norway. Plasma samples were analysed for steroid hormones by GC-MS and liver samples were analysed for environmental contaminants by GC-ECD and GC-MS. In males, we also quantified ornament traits. It was hypothesised that POPs may have endocrine disrupting effects on the local house sparrow population and can thus interfere with the steroid hormone homeostasis. Among female house sparrows, bivariate correlations revealed negative relationships between POPs and estrogens. Among male sparrows, positive relationships between dihydrotestosterone levels and PCBs were observed. In males, positive relationships were also found between steroids and beak length, and between steroids and ornamental traits such as total badge size. This was confirmed by a significant OPLS model between beak length and steroids. Although sparrows are in the mid-range trophic levels, the present study indicates that POPs may affect steroid homeostasis in house sparrows, in particular for females. For males, circulating steroid levels appears to be more associated with biometric parameters related to ornamental traits. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis, iron binding and antimicrobial properties of hexadentate 3-hydroxypyridinones-terminated dendrimers.

PubMed

Zhou, Tao; Chen, Kai; Kong, Li-Min; Liu, Mu-Song; Ma, Yong-Min; Xie, Yuan-Yuan; Hider, Robert C

2018-05-30

Macromolecular chelators have potential applications in the medical area, for instance, in treatment of iron overload-related disorders and in the treatment of external infections. In this investigation, several novel iron(III)-selective hydroxypyridinone hexadentate-terminated first and second generation dendrimeric chelators were synthesized using a convergent strategy. Their iron chelating ability was demonstrated by UV/Visible spectrometry and high resolution mass spectrometry (HRMS). The iron binding affinities were also investigated by the competition with a fluorescent iron chelator CP691. The result indicated that these dendrimers possesses a high affinity for iron with a very high pFe 3+ value, which is close to that of an isolated hexadentate unit. These dendrimeric chelators were found to exhibit inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors.

PubMed

Austin, Carol; Pettit, Simon N; Magnolo, Sharon K; Sanvoisin, Jonathan; Chen, Wenjie; Wood, Stephen P; Freeman, Lauren D; Pengelly, Reuben J; Hughes, Dallas E

2012-08-01

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

Aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith prepared via a "one-pot" process for selective extraction of ochratoxin A.

PubMed

Chen, Yiqiong; Chen, Maolin; Chi, Jinxin; Yu, Xia; Chen, Yongxuan; Lin, Xucong; Xie, Zenghong

2018-08-17

A novel aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith has been prepared with a facile "one-pot" process simultaneously via "free radical polymerization" and "thiol-ene" click reaction, and used for on-line selective extraction and practical analysis to trace ochratoxin A (OTA). By using the ternary porogenic mixture composed of water/DMF/PEG, a homogeneous polymerization mixture with POSS chemicals, acrylate-based monomers and aptamer aqueous solution was obtained, and the copolymerization of POSS chemicals, polymer monomers and aptamer aqueous solution was systematically studied. Characterizations such as the morphology, FT-IR and fluorescence spectra, mechanical stability, dynamic binding capacity, cross-reactivity and selectivity of the resultant affinity monolith were also evaluated. Attributed to the porous monolithic structure and aptamer-based affinity interaction, acceptable selective recognition and recovery yields towards trace OTA were obtained. With a 5-fold volume enrichment, the limit of detection (LOD) and limit of quantitation (LOQ) of OTA in fortified beer samples were gained at 0.025 ng/mL (S/N = 3) and 0.045 ng/mL (S/N = 10), respectively. It could be competent for the sensitive measure of actual OTA residues in real beer samples. In comparison with the previously reported strategies containing common "sol-gel" chemistry, the proposed protocol to fabricating aptamer-modified POSS-containing hybrid affinity monolith showed a simpler preparation with acceptable selectivity and higher recovery to trace OTA. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

PubMed Central

Herrmann, Claudia K.; Bukata, Lucas; Melli, Luciano; Marchesini, M. Ines; Caramelo, Julio J.

2013-01-01

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. PMID:23161032

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2011-03-01

We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)3] shows a new emission peak and the FP value of [Ru(bpy-2Gal)3] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (Kd) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)3] to TCF was confirmed by the FP measurement.

Folate/NIR 797-Conjugated Albumin Magnetic Nanospheres: Synthesis, Characterisation, and In Vitro and In Vivo Targeting Evaluation

PubMed Central

Liu, Dongfang; Liu, Peidang; Zhang, Dongsheng

2014-01-01

A practical and effective strategy for synthesis of Folate-NIR 797-conjugated Magnetic Albumin Nanospheres (FA-NIR 797-MAN) was developed. For this strategy, Magnetic Albumin Nanospheres (MAN), composed of superparamagnetic iron oxide nanoparticles (SPIONs) and bovine serum albumin (BSA), were covalently conjugated with folic acid (FA) ligands to enhance the targeting capability of the particles to folate receptor (FR) over-expressing tumours. Subsequently, a near-infrared (NIR) fluorescent dye NIR 797 was conjugated with FA-conjugated MAN for in vivo fluorescence imaging. The FA-NIR 797-MAN exhibited low toxicity to a human nasopharyngeal epidermal carcinoma cell line (KB cells). Additionally, in vitro and in vivo evaluation of the dynamic behaviour and targeting ability of FA-NIR 797-MAN to KB tumours validated the highly selective affinity of FA-NIR 797-MAN for FR-positive tumours. In summary, the FA-NIR 797-MAN prepared here exhibited great potential for tumour imaging, since the near-infrared fluorescence contrast agents target cells via FR-mediated endocytosis. The high fluorescence intensity together with the targeting effect makes FA-NIR 797-MAN a promising candidate for imaging, monitoring, and early diagnosis of cancer at the molecular and cellular levels. PMID:25188308

Synthesis and evaluation of two NIR fluorescent cyclic RGD penta-peptides for targeting integrins

NASA Astrophysics Data System (ADS)

Ye, Yunpeng; Bloch, Sharon; Xu, Baogang; Achilefu, Samuel

2006-02-01

Interest in novel RGD peptides has been increasingly growing as the interactions between RGD peptides and integrins are the basis for a variety of cellular functions and medical applications such as modulation of cell adhesion, invasion, tumor angiogenesis, and metastasis. In particular, we have been interested in novel NIR fluorescent RGD peptides as potential optical contrast agents for in vivo tumor optical imaging. Therefore, two cyclic RGD penta-peptides conjugated with a NIR fluorescent carbocyanine (Cypate), i.e. lactam-based cyclo[RGDfK(Cypate)] (1) and disulfide-containing Cypate-cyclo(CRGDC)-NH II (2), were designed and synthesized. The competitive binding assay between the purified α vβ 3 integrin and the peptide ligands using 125I-echistatin as a tracer showed that 1 had a higher receptor binding affinity (IC 50~10 -7 M) than 2 (IC 50~10 -6 M). Furthermore, the internalization of 1 in A549 cells in vitro was less than 2, as revealed by fluorescence microscopy. These results suggest that both the lactam- and disulfide-based cyclic RGD penta-peptides should be further studied structurally and functionally to elucidate the advantages of each class of compounds.

A Highly Selective and Strong Anti-Interference Host-Guest Complex as Fluorescent Probe for Detection of Amantadine by Indicator Displacement Assay.

PubMed

Zhu, Linzhao; Zhao, Zhiyong; Zhang, Xiongzhi; Zhang, Haijun; Liang, Feng; Liu, Simin

2018-04-18

Amantadine (AMA) and its derivatives are illicit veterinary drugs that are hard to detect at very low concentrations. Developing a fast, simple and highly sensitive method for the detection of AMA is highly in demand. Here, we designed an anthracyclic compound (ABAM) that binds to a cucurbit[7]uril (CB[7]) host with a high association constant of up to 8.7 × 10⁸ M −1 . The host-guest complex was then used as a fluorescent probe for the detection of AMA. Competition by AMA for occupying the cavity of CB[7] allows ABAM to release from the CB[7]-ABAM complex, causing significant fluorescence quenching of ABAM (indicator displacement assay, IDA). The linear range of the method is from 0.000188 to 0.375 μg/mL, and the detection limit can be as low as 6.5 × 10 −5 μg/mL (0.35 nM). Most importantly, due to the high binding affinity between CB[7] and ABAM, this fluorescence host-guest system shows great anti-interference capacity. Thus, we are able to accurately determine the concentration of AMA in various samples, including pharmaceutical formulations.

Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression.

PubMed

Nguyen, Minh Hong; Ojima, Yoshihiro; Sakka, Makiko; Sakka, Kazuo; Taya, Masahito

2014-10-01

Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fluorescence turn-on detection of iodide, iodate and total iodine using fluorescein-5-isothiocyanate-modified gold nanoparticles.

PubMed

Chen, Yi-Ming; Cheng, Tian-Lu; Tseng, Wei-Lung

2009-10-01

Selective turn-on fluorescence detection of I(-) was accomplished using fluorescein isothiocyanate-decorated gold nanoparticles (FITC-AuNPs). FITC molecules, which fluoresce strongly in an alkaline solution, were severely quenched when they were attached to the surface of AuNPs through their isothiocyanate group. Upon the addition of I(-), FITC molecules were detached because of I(-) adsorption on the surface of AuNPs. As a result, released FITC molecules were restored to their original fluorescence intensity. Because I(-) has a higher binding affinity to the surface of Au than do Br(-), Cl(-), or F(-), the FITC-AuNPs obviously have a higher selectivity toward I(-) than toward these other anions. Meanwhile, after IO(3)(-) was reduced to I(-) with ascorbic acid, the detection of IO(3)(-) was successfully achieved using the FITC-AuNPs. Under an optimum pH and AuNP concentration, the lowest detectable concentrations of I(-) and IO(3)(-) using this probe were 10.0 and 50.0 nM, respectively. The FITC-AuNPs provide a number of advantages, including easy preparation, selectivity, sensitivity, and low cost. This unique probe was applied to an analysis of the total iodine in edible salt and seawater.

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

PubMed

Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities[S

PubMed Central

Oswal, Dhawal P.; Balanarasimha, Madhumitha; Loyer, Jeannette K.; Bedi, Shimpi; Soman, Frances L.; Rider, S. Dean; Hostetler, Heather A.

2013-01-01

Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation. PMID:23797899

Aptamer loaded MoS2 nanoplates as nanoprobes for detection of intracellular ATP and controllable photodynamic therapy

NASA Astrophysics Data System (ADS)

Jia, Li; Ding, Lin; Tian, Jiangwei; Bao, Lei; Hu, Yaoping; Ju, Huangxian; Yu, Jun-Sheng

2015-09-01

In this work we designed a MoS2 nanoplate-based nanoprobe for fluorescence imaging of intracellular ATP and photodynamic therapy (PDT) via ATP-mediated controllable release of 1O2. The nanoprobe was prepared by simply assembling a chlorine e6 (Ce6) labelled ATP aptamer on MoS2 nanoplates, which have favorable biocompatibility, unusual surface-area-to-mass ratio, strong affinity to single-stranded DNA, and can quench the fluorescence of Ce6. After the nanoprobe was internalized into the cells and entered ATP-abundant lysosomes, its recognition to ATP led to the release of the single-stranded aptamer from MoS2 nanoplates and thus recovered the fluorescence of Ce6 at an excitation wavelength of 633 nm, which produced a highly sensitive and selective method for imaging of intracellular ATP. Meanwhile, the ATP-mediated release led to the generation of 1O2 under 660 nm laser irradiation, which could induce tumor cell death with a lysosomal pathway. The controllable PDT provided a model approach for design of multifunctional theranostic nanoprobes. These results also promoted the development and application of MoS2 nanoplate-based platforms in biomedicine.In this work we designed a MoS2 nanoplate-based nanoprobe for fluorescence imaging of intracellular ATP and photodynamic therapy (PDT) via ATP-mediated controllable release of 1O2. The nanoprobe was prepared by simply assembling a chlorine e6 (Ce6) labelled ATP aptamer on MoS2 nanoplates, which have favorable biocompatibility, unusual surface-area-to-mass ratio, strong affinity to single-stranded DNA, and can quench the fluorescence of Ce6. After the nanoprobe was internalized into the cells and entered ATP-abundant lysosomes, its recognition to ATP led to the release of the single-stranded aptamer from MoS2 nanoplates and thus recovered the fluorescence of Ce6 at an excitation wavelength of 633 nm, which produced a highly sensitive and selective method for imaging of intracellular ATP. Meanwhile, the ATP-mediated release led to the generation of 1O2 under 660 nm laser irradiation, which could induce tumor cell death with a lysosomal pathway. The controllable PDT provided a model approach for design of multifunctional theranostic nanoprobes. These results also promoted the development and application of MoS2 nanoplate-based platforms in biomedicine. Electronic supplementary information (ESI) available: Supplementary figures. See DOI: 10.1039/c5nr02224j

FRET detection of lymphocyte function–associated antigen-1 conformational extension

PubMed Central

Chigaev, Alexandre; Smagley, Yelena; Haynes, Mark K.; Ursu, Oleg; Bologa, Cristian G.; Halip, Liliana; Oprea, Tudor; Waller, Anna; Carter, Mark B.; Zhang, Yinan; Wang, Wei; Buranda, Tione; Sklar, Larry A.

2015-01-01

Lymphocyte function–associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1–specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation. PMID:25378583

Carbazole ligands as c-myc G-quadruplex binders.

PubMed

Głuszyńska, Agata; Juskowiak, Bernard; Kuta-Siejkowska, Martyna; Hoffmann, Marcin; Haider, Shozeb

2018-07-15

The interactions of c-myc G-quadruplex with three carbazole derivatives were investigated by UV-Vis spectrophotometry, fluorescence, CD spectroscopy, and molecular modeling. The results showed that a combination of carbazole scaffold functionalized with ethyl, triazole and imidazole groups resulted in stabilization of the intramolecular G-quadruplex formed by the DNA sequence derived from the NHE III 1 region of c-myc oncogene (Pu22). Binding to the G-quadruplex Pu22 resulted in the significant increase in fluorescence intensity of complexed ligands 1-3. All ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex with comparable affinity, which agrees with binding model of end-stacking on terminal G-tetrads. Copyright © 2018 Elsevier B.V. All rights reserved.

Highly sensitive and selective detection of Al(III) ions in aqueous buffered solution with fluorescent peptide-based sensor.

PubMed

In, Byunggyu; Hwang, Gi Won; Lee, Keun-Hyeung

2016-09-15

A fluorescent sensor based on a tripeptide (SerGluGlu) with a dansyl fluorophore detected selectively Al(III) among 16 metal ions in aqueous buffered solutions without any organic cosolvent. The peptide-based sensor showed a highly sensitive turn on response to aluminium ion with high binding affinity (1.84×10(4)M(-1)) in aqueous buffered solutions. The detection limit (230nM, 5.98ppb) of the peptide-based sensor was much lower than the maximum allowable level (7.41μM) of aluminium ions in drinking water demanded by EPA. The binding mode of the peptide sensor with aluminium ions was characterized using ESI mass spectrometry, NMR titration, and pH titration experiments. Copyright © 2016 Elsevier Ltd. All rights reserved.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Fluorescent recovery after photobleaching (FRAP) analysis of nuclear export rates identifies intrinsic features of nucleocytoplasmic transport.

PubMed

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-02-17

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level.

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

2012-04-01

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

Synthesis and Properties of Size-expanded DNAs: Toward Designed, Functional Genetic Systems

PubMed Central

Krueger, Andrew T.; Lu, Haige; Lee, Alex H. F.; Kool, Eric T.

2008-01-01

We describe the design, synthesis, and properties of DNA-like molecules in which the base pairs are expanded by benzo homologation. The resulting size-expanded genetic helices are called xDNA (“expanded DNA”) and yDNA (“wide DNA”). The large component bases are fluorescent, and they display high stacking affinity. When singly substituted into natural DNA, they are destabilizing because the benzo-expanded base pair size is too large for the natural helix. However, when all base pairs are expanded, xDNA and yDNA form highly stable, sequence-selective double helices. The size-expanded DNAs are candidates for components of new, functioning genetic systems. In addition, the fluorescence of expanded DNA bases makes them potentially useful in probing nucleic acids. PMID:17309194

Development of carbon dots modified fluorescent molecular imprinted Polymer@Ag/AgCl nanoparticle for hepatocellular carcinoma marker

NASA Astrophysics Data System (ADS)

Karfa, Paramita; Madhuri, Rashmi; Sharma, Prashant K.

2017-05-01

In this work, a sensitive and selective fluorescent molecularly imprinted polymer (MIP) was developed for detection of hepatocellular carcinoma (HCC) biomarker i.e. alpha feto protein (AFP) using Ag/AgCl as platform. Here, the carbon dots and Ag/AgCl nanoparticles were functionalized with vinyl groups and used as functional monomer for synthesis of AFP-imprinted polymer. The imprinted polymer shows a linear range of 3.96 ng mL-1 to 80.0 ng mL-1 with detection limit of 0.42 ng mL-1.The adsorption property of the MIP@Ag/AgCl was studied and shows the high affinity binding towards their target analyte without any cross-reactivity and false-positive or false-negative results.

Development of an aptamer-conjugated fluorescent nanoprobe for MMP2

NASA Astrophysics Data System (ADS)

Han, Myoung-Eun; Baek, Sungmin; Kim, Hyun-Jung; Lee, Jung Hwan; Ryu, Sung-Ho; Oh, Sae-Ock

2014-03-01

Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 ( K d = 5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools.

Light Armored Reconnaissance: Misunderstood and Underemployed in Deep Operations

DTIC Science & Technology

2010-04-01

then CoB 2na LA! Bn deploys in support of Operation NIMROD DANCER in Panama Dec 1989 Co D, 2d LA! Bn deploys in support of Operation JUST CAUSE in...Marine Corns Long- Range Plan (MLRP) and U.S. Marine Corps Mid-Range Plan (MMRP). An analysis of the threat discussed in these documents reveals

The Effect of School Counselors' Domain Specialization on Seniors' Milestone Completion and College Access Planning

ERIC Educational Resources Information Center

Bond, Nancy J.

2013-01-01

The purpose of this study was to determine the effect of senior high school counselors' domain specializations--academic, advanced education, career, and personal/social--from two urban high schools, on alphabetically assigned graduating seniors' with low, mid-range, and high Grade Point Averages documented college and career readiness milestone…

Judgment of Daytime Sleepiness in Self-Reported Short, Long and Midrange Sleepers

ERIC Educational Resources Information Center

Mairesse, Olivier; Neu, Daniel; Migeotte, Pierre-Francois; Pattyn, Nathalie; Hofmans, Joeri; Theuns, Peter; Cluydts, Raymond; De Valck, Elke

2012-01-01

Sleep-wake behavior, as well as sleepiness, is regulated by the joint action of an exponentially increasing drive for sleep--sleep homeostasis--and by variations in sleep propensity due to a biological circadian oscillator. However, large inter-individual differences remain. Short and long sleepers have been known to differ in the amount of…

Development of a H2 O2 -sensitive quantum dots-based fluorescent sandwich ELISA for sensitive detection of bovine β-lactoglobulin by monoclonal antibody.

PubMed

He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

2018-01-01

Bovine β-lactoglobulin (BLG) is the major allergen in cows' milk, and the specific epitope plays a key role in food allergy. Developing a method specifically bind to the IgE epitope is necessary for testing BLG and its allergenic residues. The monoclonal antibody (1G9) specific to the IgE linear epitope for BLG was identified as high affinity and specificity. Based on 1G9, a sensitive fluorescent sandwich enzyme-linked immunosorbent assay (sELISA) was successfully developed using catalase-mediated fluorescence quenching of thiolated CdTe quantum dots in the presence of hydrogen peroxide as fluorescent signal output. The fluorescent sELISA showed high sensitivity and specificity, the limit of detection was 0.49 ng mL -1 , which was 16-fold lower than horseradish peroxidase (HRP)-based sELISA. The linear range for BLG detection were 125-4000 ng mL -1 (r = 0.9939) and 0.48-62.5 ng mL -1 (r = 0.9919). The recoveries and coefficients of variation were 94.25-109.83% and 4.38-20.29%, respectively. Allergenic residues were also detected in hydrolysed infant formulas. The results of fluorescent sELISA showed good performance as HRP-based sELISA and commercial sELISA kit. This proposed fluorescent sELISA could be employed to detect BLG and its allergenic residues in food with highly sensitivity, reliability, and recovery. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1994-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a polycationic chain of at least two positive charges. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

NASA Astrophysics Data System (ADS)

Perez-Gonzalez, Cibran; Lafontaine, Daniel; Penedo, J.

2016-08-01

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

PubMed Central

Perez-Gonzalez, Cibran; Lafontaine, Daniel A.; Penedo, J. Carlos

2016-01-01

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques. PMID:27536656

Affinity study on bovine serum albumin's peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

NASA Astrophysics Data System (ADS)

Yuan, Ming; Li, Wanrong; Yang, Mingming; Huang, Xiufeng; Bai, Zhijun; Liu, Yushuang; Cai, Weijun; Wang, Yuqin; Zhang, Feng

2017-09-01

It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1 > Ka-S2 >> Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.

Interaction of thionine with triple-, double-, and single-stranded RNAs.

PubMed

Lozano, Héctor J; García, Begoña; Busto, Natalia; Leal, José M

2013-01-10

The interaction of thionine with triple, double, and single RNA helices has been fully characterized by thermodynamic and kinetic methods. The nature of the interaction of thionine with the synthetic polynucleotides poly(rU), poly(rA)·poly(rU), and poly(rA)·2poly(rU) has been studied at pH = 7.0 and 25 °C by UV absorbance, fluorescence, circular dichroism spectroscopy, viscometry, differential scanning calorimetry, and T-jump kinetic measurements. The results show that at I = 0.1 M thionine binds to a single poly(rU) strand, destabilizes the poly(rA)·2poly(rU) triplex by external binding, and intercalates into poly(rA)·poly(rU) with similar affinity to the thionine/DNA intercalated complex (Paul, P.; Kumar, G. S. J. Fluoresc. 2012, 22, 71-80). On the other hand, the differential scanning calorimetry measurements performed with thionine display a point in which the heat capacity remains unaltered, revealing the equilibrium of isothermal denaturation: thionine/poly(rA)·2poly(rU) + thionine ⇌ thionine/poly(rA)·poly(rU) + thionine/poly(rU), an outcome supported by the other techniques used. The denaturation equilibrium constant, K(D) (25 °C) = 522 M(-1), was evaluated from the affinity with the single, duplex, and triplex RNA.

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe.

PubMed

Shvadchak, Volodymyr V; Falomir-Lockhart, Lisandro J; Yushchenko, Dmytro A; Jovin, Thomas M

2011-04-15

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins.

PubMed

Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2018-06-15

Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α 1 -acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222. Copyright © 2018 Elsevier B.V. All rights reserved.

Self-aggregation of bio-surfactants within ionic liquid 1-ethyl-3-methylimidazolium bromide: A comparative study and potential application in antidepressants drug aggregation.

PubMed

Banjare, Manoj Kumar; Behera, Kamalakanta; Kurrey, Ramsingh; Banjare, Ramesh Kumar; Satnami, Manmohan L; Pandey, Siddharth; Ghosh, Kallol K

2018-06-15

Aggregation behavior of bio-surfactants (BS) sodium cholate (NaC) and sodium deoxycholate (NaDC) within aqueous solution of ionic liquid (IL) 1-ethyl-3-methylimidazolium bromide [Emim][Br] has been investigated using surface tension, conductivity, steady state fluorescence, FT-IR and dynamic light scattering (DLS) techniques. Various interfacial and thermodynamic parameters are determined in the presence of different wt% of IL [Emim][Br]. Information regarding the local microenvironment and size of the aggregates is obtained from fluorescence and DLS, respectively. FT-IR spectral response is used to reveal the interactions taking place within aqueous NaC/NaDC micellar solutions. It is noteworthy to mention that increasing wt% of [Emim][Br] results in an increase in the spontaneity of micelle formation and the hydrophilic IL shows more affinity for NaC as compared to NaDC. Further, the micellar solutions of BS-[Emim][Br] are utilized for studying the aggregation of antidepressants drug promazine hydrochloride (pH). UV-vis spectroscopic investigation reveals interesting outcomes and the results show changes in spectral absorbance of PH drug on the addition of micellar solution (BS-[Emim][Br]). Highest binding affinity and most promising activity are shown for NaC as compared to NaDC. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorescence and NMR investigations in the ligand binding properties of adenylate kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinstein, J.; Vetter, I.R.; Schlichting, I.

A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand {alpha},{omega}-di((3{prime} or 2{prime})-O-(N-methyl-anthraniloyl)adenosine-5{prime}) pentaphosphate (MAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 {times} 10{sup {minus}9} to 5 {times} 10{sup {minus}2} M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors ismore » proposed and applied to these data. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. Together with the results obtained by Tian et al on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.« less

Inferring diffusion dynamics from FCS in heterogeneous nuclear environments.

PubMed

Tsekouras, Konstantinos; Siegel, Amanda P; Day, Richard N; Pressé, Steve

2015-07-07

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The effects of electromagnetic pulses (EMP) on the bioactivity of insulin and a preliminary study of mechanism.

PubMed

Chen, Yong Bin; Li, Jing; Qi, Yuhong; Miao, Xia; Zhou, Yongchun; Ren, Dongqing; Guo, G Z

2010-01-01

To investigate the effects of electromagnetic pulse (EMP) exposure on the bioactivity of insulin and a preliminary mechanism for these effects. A tapered parallel plate Gigahertz Transverse Electromagnetic (GTEM) cell with a flared rectangular coaxial transmission line was used to expose the insulin solution to EMP. Concurrent sham-exposed insulin solutions were used as a control. The effect of EMP-exposed insulin on fasting blood glucose levels of type I diabetes model mice, the effect of EMP on binding affinity between insulin and its receptor and the effect of EMP on insulin's fluorescence intensity were detected, respectively. (i) After EMP exposure, compared with sham-exposed insulin, the bioactivity of insulin in decreasing fasting blood glucose levels in type I diabetes model mice was reduced significantly (p = 0.023). (ii) Compared with sham-exposed insulin group, the percentage fluorescein isothiocyannate (FITC) labelling of HL-7702 cells was significantly reduced in the EMP-exposed insulin group (22.7-13.8%, respectively). (iii) Compared with sham-exposed insulin, the fluorescence intensity was significantly reduced in EMP-exposed insulin (p < 0.001). EMP exposure significantly decreased the bioactivity of insulin to reduce the blood glucose levels in type I diabetic mice. This could be due to a decreased binding affinity between insulin and its receptor. This mechanism could involve an alteration of insulin's' conformation caused by EMP exposure.

A novel fluorescent aptasensor based on hairpin structure of complementary strand of aptamer and nanoparticles as a signal amplification approach for ultrasensitive detection of cocaine.

PubMed

Emrani, Ahmad Sarreshtehdar; Danesh, Noor Mohammad; Ramezani, Mohammad; Taghdisi, Seyed Mohammad; Abnous, Khalil

2016-05-15

Cocaine is one of the most commonly misused stimulant which could influence the central nervous system. In this study, a fluorescent aptamer-based sensor (aptasensor) was designed for sensitive and selective detection of cocaine, based on hairpin structure of complementary strand of aptamer (CS), target-induced release of aptamer (Apt) from CS and two kinds of nanoparticles, including silica nanoparticles (SNPs) coated with streptavidin and gold nanoparticles (AuNPs). The designed aptasensor acquires characteristics of AuNPs such as unique optical properties and large surface area, SNPs as amplifiers of fluorescence intensity, higher affinity of Apt toward its target relative to its CS, and finally the hairpin structure of CS that brings the fluorophore (FAM) to close proximity to the surface of SNPs. In the absence of cocaine, FAM is in close proximity to the surface of AuNPs, resulting in a weak fluorescence emission. In the presence of target, FAM comes to close proximity to the surface of SNPs because of the formation of hairpin structure of CS, leading to a very strong fluorescence emission. The fabricated fluorescent aptasensor exhibited a good selectivity toward cocaine with a limit of detection (LOD) as low as 209 pM. Moreover, the designed aptasensor was successfully utilized to detect cocaine in serum with a LOD as low as 293 pM. Copyright © 2015 Elsevier B.V. All rights reserved.

An ATMND/SGI based label-free and fluorescence ratiometric aptasensor for rapid and highly sensitive detection of cocaine in biofluids.

PubMed

Wang, Jiamian; Song, Jie; Wang, Xiuyun; Wu, Shuo; Zhao, Yanqiu; Luo, Pinchen; Meng, Changgong

2016-12-01

A label-free ratiometric fluorescence aptasensor has been developed for the rapid and sensitive detection of cocaine in complex biofluids. The fluorescent aptasensor is composed of a non-labeled GC-38 cocaine aptamer which serves as a basic sensing unit and two fluorophores, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and SYBR Green I (SGI) which serves as a signal reporter and a build-in reference, respectively. The detection principle is based on a specific cocaine mediated ATMND displacement reaction and the corresponding change in the fluorescence ratio of ATMND to SGI. Due to the high affinity of the non-labeled aptamer, the good precision originated from the ratiometric method, and the good fluorescence quantum yield of the fluorophore, the aptasensor shows good analytical performance with respect to cocaine detection. Under optimal conditions, the aptasensor shows a linear range of 0.10-10μM and a low limit of detection of 56nM, with a fast response of 20s. The low limit of detection is comparable to most of the fluorescent aptasensors with signal amplification strategies and much lower than all of the unamplified cocaine aptasensors. Practical sample analysis in a series of complex biofluids, including urine, saliva and serum, also indicates the good precision, stability, and high sensitivity of the aptasensor, which may have great potential for the point-of-care screening of cocaine in complex biofluids. Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular zinc distribution in mitochondria, ER and the Golgi apparatus

PubMed Central

Lu, Qiping; Haragopal, Hariprakash; Slepchenko, Kira G; Stork, Christian; Li, Yang V

2016-01-01

Zinc (Zn2+) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis. PMID:27186321

The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate.

PubMed Central

Roberts, D; Kellett, G L

1979-01-01

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state. Images Fig. 5. Fig. 8. PMID:160791

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

PubMed Central

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

2015-01-01

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

PubMed

Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

2015-07-07

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Glowing locked nucleic acids: brightly fluorescent probes for detection of nucleic acids in cells.

PubMed

Østergaard, Michael E; Cheguru, Pallavi; Papasani, Madhusudhan R; Hill, Rodney A; Hrdlicka, Patrick J

2010-10-13

Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2'-N-(pyren-1-yl)carbonyl-2'-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (Φ(F) = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

PubMed

Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

2015-03-03

A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

Synthesis of highly water-soluble fluorescent conjugated glycopoly(p-phenylene)s for lectin and Escherichia coli.

PubMed

Xue, Cuihua; Jog, Sonali P; Murthy, Pushpalatha; Liu, Haiying

2006-09-01

Two facile, convenient, and versatile synthetic approaches are used to covalently attach carbohydrate residues to conjugated poly(p-phenylene)s (PPPs) for highly water-soluble PPPs bearing alpha-mannopyranosyl and beta-glucopyranosyl pendants (polymers A and B), which highly fluoresce in phosphate buffer (pH 7.0). The post-polymerization functionalization approach is to treat bromo-bearing PPP (polymer 1) with 1-thiolethyl-alpha-D-mannose tetraacetate or 1-thiol-beta-D-glucose tetraacetate in THF solution in the presence of K(2)CO(3) at room temperature through formation of thioether bridges, affording polymer 2a or 2b. The prepolymerization functionalization approach is to polymerize a well-defined sugar-carrying monomer, affording polymer 2a. Polymers 2a and 2b were deacetylated under Zemplén conditions in methanol and methylene chloride containing sodium methoxide, affording polymers A and B, respectively. The multivalent display of carbohydrates on the fluorescent conjugated glycopolymer overcomes the characteristic low binding affinity of the individual carbohydrates to their receptor proteins. Titration of concanavalin A (Con A) to alpha-mannose-bearing polymer A resulted in significant fluorescent quenching of the polymer with Stern-Volmer quenching constant of 4.5 x 10(7). Incubation of polymer A with Escherichia coli (E. coli) lead to formation of fluorescently stained bacterial clusters. Beta-glucose-bearing polymer B displayed no response to Con A and E. coli.

In vivo detection of amyloid plaques in the mouse brain using the near-infrared fluorescence probe THK-265.

PubMed

Okamura, Nobuyuki; Mori, Masanori; Furumoto, Shozo; Yoshikawa, Takeo; Harada, Ryuichi; Ito, Satoshi; Fujikawa, Yosuke; Arai, Hiroyuki; Yanai, Kazuhiko; Kudo, Yukitsuka

2011-01-01

Noninvasive detection of amyloid-β (Aβ) deposits in the brain would be beneficial for an early and presymptomatic diagnosis of Alzheimer's disease (AD). We developed THK-265 as a candidate near-infrared fluorescence (NIRF) probe for the in vivo detection of amyloid deposits in the brain. The maximal emission wavelength of THK-265 was greater than 650nm and it showed high quantum yield and molar absorption coefficients. A fluorescence binding assay showed its high binding affinity to Aβ fibrils (Kd = 97 nM). THK-265 clearly stained amyloid plaques in AD neocortical brain sections and showed a moderate log p value (1.8). After intravenous administration of THK-265 in amyloid-β protein precursor (AβPP) transgenic mice, amyloid deposits in the brain were clearly labeled with THK-265. Furthermore, in vivo NIRF imaging demonstrated significantly higher fluorescence intensity in the brains of AβPP transgenic mice than in those of wild-type mice. As THK-265 showed profound hyperchromic effect upon binding to Aβ fibrils, good discrimination between AβPP transgenic and wild-type mice was demonstrated even early after THK-265 administration. Furthermore, the fluorescence intensity of THK-265 correlated with amyloid plaque burden in the brains of AβPP transgenic mice. These findings strongly support the usefulness of THK-265 as an NIRF imaging probe for the noninvasive measurement of brain amyloid load.

Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

DOE PAGES

Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

2015-12-30

Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

Facile synthesis of Fe3O4/g-C3N4/HKUST-1 composites as a novel biosensor platform for ochratoxin A.

PubMed

Hu, Shuisheng; Ouyang, Wenjun; Guo, Longhua; Lin, Zhenyu; Jiang, Xiaohua; Qiu, Bin; Chen, Guonan

2017-06-15

A fluorescent biosensor for ochratoxin A was fabricated on the basis of a new nanocomposite (Fe 3 O 4 /g-C 3 N 4 /HKUST-1 composites). Fe 3 O 4 /g-C 3 N 4 /HKUST-1 was synthesized in this work for the first time, which combined HKUST-1 with g-C 3 N 4 to improve its chemical stability. Fe 3 O 4 /g-C 3 N 4 /HKUST-1 composites have strong adsorption capacity for dye-labeled aptamer and are able to completely quench the fluorescence of the dye through the photoinduced electron transfer (PET) mechanism. In the presence of ochratoxin A (OTA), it can bind with the aptamer with high affinity, causing the releasing of the dye-labeled aptamer from the Fe 3 O 4 /g-C 3 N 4 /HKUST-1 and therefore results in the recovery of fluorescence. The fluorescence intensity of the biosensor has a linear relationship with the OTA concentration in the range of 5.0-160.0ng/mL. The LOD of sensor is 2.57ng/mL (S/N=3). This fluorescence sensor based on the Fe 3 O 4 /g-C 3 N 4 /HKUST-1 composites has been applied to detect OTA in corn with satisfying results. Copyright © 2016. Published by Elsevier B.V.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing.

PubMed

Morozov, Giora I; Zhao, Huaying; Mage, Michael G; Boyd, Lisa F; Jiang, Jiansheng; Dolan, Michael A; Venna, Ramesh; Norcross, Michael A; McMurtrey, Curtis P; Hildebrand, William; Schuck, Peter; Natarajan, Kannan; Margulies, David H

2016-02-23

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis.

PubMed

Milles, Sigrid; Lemke, Edward A

2014-07-07

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of methoxy-X04 derivatives and their evaluation in Alzheimer's disease pathology.

PubMed

Boländer, Alexander; Kieser, Daniel; Scholz, Christoph; Heyny-von Haußen, Roland; Mall, Gerhard; Goetschy, Valérie; Czech, Christian; Schmidt, Boris

2014-01-01

Alzheimer's disease is characterized by two notorious protein aggregates in the brain: extracellular senile plaques mainly consisting of amyloid-β peptides and tau-protein-derived intracellular paired helical filaments. The diagnosis of Alzheimer's disease is impaired by insufficient sensitivity and specificity of diagnostic methods to visualize these pathological hallmarks over all disease stages. The established fluorescence marker methoxy-X04 stains plaques, tau tangles and amyloid-derived angiopathies with good specificity, yet it is limited by slow elimination in vivo. Since the need for new markers is high, we prepared methoxy-X04 derivatives and evaluated their potential as imaging agents in Alzheimer's disease pathology. In this study, we describe an improved synthesis for methoxy-X04 and its derivatives and their affinity determination for the respective protein targets by immunohistology and a displacement assay. This resulted in the identification of new derivatives of methoxy-X04 with improved binding affinity.

Modular microfluidics for point-of-care protein purifications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Millet, L. J.; Lucheon, J. D.; Standaert, R. F.

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

Modular microfluidics for point-of-care protein purifications.

PubMed

Millet, L J; Lucheon, J D; Standaert, R F; Retterer, S T; Doktycz, M J

2015-04-21

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE PAGES

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.; ...

2016-02-11

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

Modular microfluidics for point-of-care protein purifications

DOE PAGES

Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; ...

2015-01-01

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

Binding of resveratrol with sodium caseinate in aqueous solutions.

PubMed

Acharya, Durga P; Sanguansri, Luz; Augustin, Mary Ann

2013-11-15

The interaction between resveratrol (Res) and sodium caseinate (Na-Cas) has been studied by measuring fluorescence quenching of the protein by resveratrol. Quenching constants were determined using Stern-Volmer equation, which suggests that both dynamic and static quenching occur between Na-Cas and Res. Binding constants for the complexation between Na-Cas and Res were determined at different temperatures. The large binding constants (3.7-5.1×10(5)M(-1)) suggest that Res has strong affinity for Na-Cas. This affinity decreases as the temperature is raised from 25 to 37°C. The binding involves both hydrogen bonding and hydrophobic interaction, as suggested by negative enthalpy change and positive entropy change for the binding reaction. The present study indicates that Na-Cas, a common food protein, may be used as a carrier of Res, a bioactive polyphenol which is insoluble in both water and oils. Copyright © 2013 Elsevier Ltd. All rights reserved.

Longleaf pine bud development: influence of seedling nutrition

Treesearch

J. P. Barnett; D. P. Jackson; R. K. Dumroese

2010-01-01

A subset of seedlings from a larger study (Jackson and others 2006, 2007) were selected and evaluated for two growing seasons to relate bud development, and root-collar diameter (RCD), and height growth with three nursery fertilization rates. We chose seedlings in the 0.5 (lowest), 2.0 (mid-range), and 4.0 (highest) mg of nitrogen per seedling treatments. Buds moved...

Full Hybrid: Cruising

Science.gov Websites

1 At speeds above mid-range, both the engine and electric motor are used to propel the vehicle. The gasoline engine provides power to the drive-train directly and to the electric motor via the generator. Go , generator, power split device, and electric motor visible. The car is moving. There are blue arrows flowing

High Performance Flexible Actuator of Urchin-Like ZnO Nanostructure/Polyvinylenefluoride Hybrid Thin Film with Graphene Electrodes for Acoustic Generator and Analyzer.

PubMed

Cheong, Oug Jae; Lee, James S; Kim, Jae Hyun; Jang, Jyongsik

2016-05-01

A bass frequency response enhanced flexible polyvinylidene fluoride (PVDF) based thin film acoustic actuator is successfully fabricated. High concentrations of various zinc oxide (ZnO) is embedded in PVDF matrix, enhancing the β phase content and the dielectric property of the composite thin film. ZnO acts as a nucleation agent for the crystallization of PVDF. A chemical vapor deposition grown graphene is used as electrodes, enabling high electron mobility for the distortion free acoustic signals. The frequency response of the fabricated acoustic actuator is studied as a function of the film thickness and filler content. The optimized film has a thickness of 80 μm with 30 wt% filler content and shows 72% and 42% frequency response enhancement in bass and midrange compared to the commercial PVDF, respectively. Also, the total harmonic distortion decreases to 82% and 74% in the bass and midrange regions, respectively. Furthermore, the composite film shows a promising potential for microphone applications. Most of all, it is demonstrated that acoustic actuator performance is strongly influenced by degree of PVDF crystalline. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Binding Specificity of Two PBPs in the Yellow Peach Moth Conogethes punctiferalis (Guenée)

PubMed Central

Ge, Xing; Ahmed, Tofael; Zhang, Tiantao; Wang, Zhenying; He, Kanglai; Bai, Shuxiong

2018-01-01

Pheromone binding proteins (PBPs) play an important role in olfaction of insects by transporting sex pheromones across the sensillum lymph to odorant receptors. To obtain a better understanding of the molecular basis between PBPs and semiochemicals, we have cloned, expressed, and purified two PBPs (CpunPBP2 and CpunPBP5) from the antennae of Conogethes punctiferalis. Fluorescence competitive binding assays were used to investigate binding affinities of CpunPBP2 and CpunPBP5 to sex pheromone and volatiles. Results indicate both CpunPBP2 and CpunPBP5 bind sex pheromones E10-16:Ald, Z10-16:Ald and hexadecanal with higher affinities. In addition, CpunPBP2 and CpunPBP5 also could bind some odorants, such as 1-tetradecanol, trans-caryopyllene, farnesene, and β-farnesene. Homology modeling to predict 3D structure and molecular docking to predict key binding sites were used, to better understand interactions of CpunPBP2 and CpunPBP5 with sex pheromones E10-16:Ald and Z10-16:Ald. According to the results, Phe9, Phe33, Ser53, and Phe115 were key binding sites predicted for CpunPBP2, as were Ser9, Phe12, Val115, and Arg120 for CpunPBP5. Binding affinities of four mutants of CpunPBP2 and four mutants of CpunPBP5 with the two sex pheromones were investigated by fluorescence competitive binding assays. Results indicate that single nucleotides mutation may affect interactions between PBPs and sex pheromones. Expression levels of CpunPBP2 and CpunPBP5 in different tissues were evaluated using qPCR. Results show that CpunPBP2 and CpunPBP5 were largely amplified in the antennae, with low expression levels in other tissues. CpunPBP2 was expressed mainly in male antennae, whereas CpunPBP5 was expressed mainly in female antennae. These results provide new insights into understanding the recognition between PBPs and ligands. PMID:29666585

The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

PubMed

Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

2016-04-01

Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

PubMed

Ries, Jonas; Yu, Shuizi Rachel; Burkhardt, Markus; Brand, Michael; Schwille, Petra

2009-09-01

Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

PubMed

Abdelhamid, Mohamed A A; Ikeda, Takeshi; Motomura, Kei; Tanaka, Tatsuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

2016-11-01

We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport*

PubMed Central

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-01-01

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level. PMID:22190681

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

PubMed Central

Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

2016-01-01

Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics. PMID:27152703

Calcium-Dependent Energetics of Calmodulin Domain Interactions with Regulatory Regions of the Ryanodine Receptor Type 1 (RyR1)

PubMed Central

Newman, Rhonda A.; Sorensen, Brenda R.; Kilpatrick, Adina M.; Shea, Madeline A.

2014-01-01

Calmodulin (CaM) plays a vital role in calcium homeostasis by allosterically modulating intracellular calcium channels including the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1). Apo (calcium-free) CaM activates hRyR1 while calcium-saturated CaM inhibits it. Two CaM-binding regions (residues 1975–1999 and 3614–3643) identified in each RyR1 monomer were proposed to allow CaM to bridge adjacent RyR1 subunits. We explored the distinct roles of CaM domains by using fluorescence anisotropy to determine the affinity of CaM1–148 (full-length), CaM1–80 (N-domain) and CaM76–148 (C-domain) for peptides encompassing hRyR1 residues 1975–1999 or 3614–3643. Both CaM1–148 and CaM76–148 associated in a calcium-independent manner with similar affinities for hRyR1(3614–3643)p while CaM1–80 required calcium and bound ~250-fold more weakly. Association of CaM1–148, CaM1–80 and CaM76–148 with hRyR1(1975–1999)p was much less favorable than with hRyR1(3614–3643)p; differences between the two CaM domains were smaller. Equilibrium calcium titrations monitored by steady-state fluorescence demonstrated that both hRyR1 peptides increased the calcium-binding affinity of both CaM domains. These thermodynamic properties support a prior model in which the CaM C-domain associates with RyR1(3614–3643) at low levels of calcium, positioning CaM to rapidly respond to calcium efflux. However, the affinity of the N-domain of CaM for hRyR1(1975–1999)p is insufficient to explain a model in which CaM bridges adjacent RyR1 subunits within the tetramer. This indicates that other protein factors or properties of the tertiary or quaternary structure of hRyR1 contribute to the energetics of CaM-mediated regulation. PMID:25145833

Ultrasensitive detection of lead (II) based on fluorescent aptamer-functionalized carbon nanotubes.

PubMed

Taghdisi, Seyed Mohammad; Emrani, Somayeh Sarreshtehdar; Tabrizian, Kaveh; Ramezani, Mohammad; Abnous, Khalil; Emrani, Ahmad Sarreshtehdar

2014-05-01

Lead contamination is a serious environmental problem with toxic effects in human. Here, we developed a simple and sensitive sensing method employing ATTO 647N/aptamer-SWNT ensemble for detection of Pb(2+). This method is based on the super quenching capability of single-walled carbon nanotubes (SWNTs), high affinity of the aptamer toward Pb(2+) and different propensities of ATTO 647N-aptamer and ATTO 647N-aptamer/Pb(2+) complex for adsorption on SWNTs. In the absence of Pb(2+), the fluorescence of ATTO 647N-aptamer is efficiently quenched by SWNTs. Upon addition of Pb(2+), the aptamer binds to its target, leading to the formation of a G-quadruplex/Pb(2+) complex and does not interact with SWNTs and ATTO 647N-aptamer starts fluorescing. This sensor exhibited a high selectivity toward Pb(2+) and a limit of detection (LOD) as low as 0.42 nM was obtained. Also this sensor could be applied for detection of Pb(2+) ions in tap water and biological sample like serum with high sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy.

PubMed

Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

2014-10-10

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding. Copyright © 2014 Elsevier Inc. All rights reserved.

A simple system for the identification of fluorescent dyes capable of reporting differences in secondary structure and hydrophobicity among amyloidogenic protein oligomers

NASA Astrophysics Data System (ADS)

Yates, Emma

2012-02-01

Thioflavin T and Congo Red are fluorescent dyes that are commonly used to identify the presence of amyloid structures, ordered protein aggregates. Despite the ubiquity of their use, little is known about their mechanism of interaction with amyloid fibrils, or whether other dyes, whose photophysics indicate that they may be more responsive to differences in macromolecular secondary structure and hydrophobicity, would be better suited to the identification of pathologically relevant oligomeric species in amyloid diseases. In order to systematically address this question, we have designed a strategy that discretely introduces differences in secondary structure and hydrophobicity amidst otherwise identical polyamino acids. This strategy will enable us to quantify and compare the affinities of Thioflavin T, Congo Red, and other, incompletely explored, fluorescent dyes for different secondary structural elements and hydrophobic motifs. With this information, we will identify dyes that give the most robust and quantitative information about structural differences among the complex population of oligomeric species present along an aggregation pathway between soluble monomers and amyloid fibrils, and correlate the resulting structural information with differential oligomeric toxicity.

Modulated photophysics of a cationic DNA-staining dye inside protein bovine serum albumin: Study of binding interaction and structural changes of protein

NASA Astrophysics Data System (ADS)

Samanta, Anuva; Jana, Sankar; Ray, Debarati; Guchhait, Nikhil

2014-03-01

The binding affinity of cationic DNA-staining dye, propidium iodide, with transport protein, bovine serum albumin, has been explored using UV-vis absorption, fluorescence, and circular dichroism spectroscopy. Steady state and time resolved fluorescence studies authenticate that fluorescence quenching of bovine serum albumin by propidium iodide is due to bovine serum albumin-propidium iodide complex formation. Thermodynamic parameters obtained from temperature dependent spectral studies cast light on binding interaction between the probe and protein. Site marker competitive binding has been encountered using phenylbutazone and flufenamic acid for site I and site II, respectively. Energy transfer efficiency and distance between bovine serum albumin and propidium iodide have been determined using Förster mechanism. Structural stabilization or destabilization of protein by propidium iodide has been investigated by urea denaturation study. The circular dichroism study as well as FT-IR measurement demonstrates some configurational changes of the protein in presence of the dye. Docking studies support the experimental data thereby reinforcing the binding site of the probe to the subdomain IIA of bovine serum albumin.

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

PubMed

Buijs; Hlady

1997-06-01

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria.

PubMed

Grünstein, Dan; Maglinao, Maha; Kikkeri, Raghavendra; Collot, Mayeul; Barylyuk, Konstantin; Lepenies, Bernd; Kamena, Faustin; Zenobi, Renato; Seeberger, Peter H

2011-09-07

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

PubMed

Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

2016-04-01

Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

PubMed Central

Comte, M; Malnoë, A; Cox, J A

1986-01-01

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5. Images Fig. 2. PMID:3814096

The solvatochromic effects of side chain substitution on the binding interaction of novel tricarbocyanine dyes with human serum albumin.

PubMed

Beckford, Garfield; Owens, Eric; Henary, Maged; Patonay, Gabor

2012-04-15

The effects of solvatochromism on protein-ligand interactions have been studied by absorbance and near-infrared laser induced fluorescence (NIR-LIF) spectroscopy. The utility of three novel classes of cyanine dyes designed for this purpose illustrates that the affinity interactions of ligands at the hydrophobic binding pockets of Human Serum Albumin (HSA) are not only dependent on the overall hydrophobic characteristics of the molecules but are highly influenced by the size of the ligands as well. Whereas changes to the chromophore moiety exhibited slight to moderate changes to the hydrophobic nature of these molecules, substitution at the alkyl indolium side chain has enabled us to vary the binding affinity towards serum albumin. Substitution at the indolium side chain among an ethyl to butyl group results in improved binding characteristics and an almost three-fold increase in affinity constant. In addition, replacement of the ethyl side chain with a phenylpropyl group also yielded unique solvotachromic patterns such as increased hydrophobicity and subsequent biocompatibility with the HSA binding regions. Ligand interaction was however inhibited by steric hindrance associated with the bulky phenyl ring system thus affecting the increased binding that could be realized from the improved hydrophobic nature of the molecules. This characteristic change in binding affinity is of potential interest to developing a methodology which reveals information on the hydrophobic character and steric specificity of the binding cavities. Copyright © 2012 Elsevier B.V. All rights reserved.

Fluorescence Imaging Study of Extracellular Zinc at the Hippocampal Mossy Fiber Synapse

PubMed Central

Bastian, Chinthasagar; Li, Yang V

2010-01-01

Although synaptically-released, vesicular Zn2+ has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn2+ release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn2+ at the mossy fiber synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn2+ indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green (KD Zn2+ ~ 1 μM), Zinpyr-4 (KD Zn2+ ~ 1 nM) and FluoZin-3 (KD Zn2+ ~ 15 nM), chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca2+ (KD Ca2+ ~ 100 μM) which was present in the extracellular medium ([Ca2+]o > 2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically-released Zn2+ in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn2+ onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn2+ contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system. PMID:17485170

Preclinical Comparison of Near-Infrared-Labeled Cetuximab and Panitumumab for Optical Imaging of Head and Neck Squamous Cell Carcinoma

PubMed Central

Day, Kristine E.; Sweeny, Larissa; Kulbersh, Brian; Zinn, Kurt R.; Rosenthal, Eben L.

2014-01-01

Purpose: Though various targets have been proposed and evaluated, no agent has yet been investigated in a clinical setting for head and neck cancer. The present study aimed to compare two fluorescently labeled anti-epidermal growth factor receptor (EGFR) antibodies for detection of head and neck squamous cell carcinoma (HNSCC). Procedures: Antigen specificities and in vitro imaging of the fluorescently labeled anti-EGFR antibodies were performed. Next, immunodeficient mice (n=22) bearing HNSCC (OSC-19 and SCC-1) tongue tumors received systemic injections of cetuximab-IRDye800CW, panitumumab-IRDye800CW, or IgG-IRDye800CW (a nonspecific control). Tumors were imaged and resected using two near-infrared imaging systems, SPY and Pearl. Fluorescent lymph nodes were also identified, and all resected tissues were sent for pathology. Results: Panitumumab-IRDye800CW and cetuximab-IRDye800CW had specific and high affinity binding for EGFR (KD=0.12 and 0.31 nM, respectively). Panitumumab-IRDye800CW demonstrated a 2-fold increase in fluorescence intensity compared to cetuximab-IRDye800CW in vitro. In vivo, both fluorescently labeled antibodies produced higher tumor-to-background ratios compared to IgG-IRDye800CW. However, there was no significant difference between the two in either cell line or imaging modality (OSC-19: p=0.08 SPY, p=0.48 Pearl; SCC-1: p=0.77 SPY, p=0.59 Pearl; paired t tests). Conclusions: There was no significant difference between the two fluorescently labeled anti-EGFR monoclonal antibodies in murine models of HNSCC. Both cetuximab and panitumumab can be considered suitable targeting agents for fluorescent intraoperative detection of HNSCC. PMID:23715932

EGFR-directed Affibody for fluorescence-guided glioma surgery: time-dose analysis (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ribeiro de Souza, Ana Luiza; Marra, Kayla; Gunn, Jason R.; Elliott, Jonathan T.; Samkoe, Kimberley S.; Paulsen, Keith D.; Draney, Daniel R.; Feldwisch, Joachim

2016-03-01

The key to fluorescence guided surgical oncology is the ability to create specific contrast between normal and glioma tissue. The blood brain barrier that limits the delivery of substances to the normal brain is broken in tumors, allowing accumulation of agents in the tumor interior. However, for a clinical success, imaging agents should be in the infiltrative edges to minimize the resection of normal brain while enable the removal of tumor. The aberrant overexpression and/or activation of EGFR is associated with many types of cancers, including glioblastoma and the injection of a fluorescent molecule targeted to these receptors would improve tumor contrast during fluorescence guided surgery. Affibody molecules have intentional medium affinity and high potential specificity, which are the desirable features of a good surgical imaging agent. The aim of this study was evaluate the brain/glioma uptake of ABY029 labeled with near-infrared dye IRDye800CW after intravenous injection. Rats were either inoculated with orthotopic implantations of U251 human glioma cell line or PBS (shams control) in the brain. The tumors were allowed to grow for 2-3 weeks before carrying out fluorescent tracer experiments. Fluorescent imaging of ex vivo brain slices from rats was acquired at different time points after infection of fluorescently labeled EGFR-specific affibody to verify which time provided maximal contrast tumor to normal brain. Although the tumor was most clearly visualized after 1h of IRDye800CW-labeled ABY029 injection, the tumor location could be identified from the background after 48h. These results suggest that the NIR-labeled affibody examined shows excellent potential to increase surgical visualization for confirmed EGFR positive tumors.

The trehalose/maltose-binding protein as the sensitive element of a glucose biosensor

NASA Astrophysics Data System (ADS)

Fonin, A. V.; Povarova, O. I.; Staiano, M.; D'Auria, S.; Turoverov, K. K.; Kuznetsova, I. M.

2014-08-01

The promising direction of the development of a modern glucometer is the construction of sensing element on the basis of stained (dyed) protein which changes its fluorescence upon glucose binding. One of the proteins that can be used for this purpose is the D-trehalose/D-maltose-binding protein (TMBP) from the thermophilic bacteria Thermococcus litoralis. We investigated the physical-chemical properties of the protein and evaluated its stability to the denaturing action of GdnHCl and heating. It was confirmed that TMBP is an extremely stable protein. In vivo, the intrinsic ligands of TMBP are trehalose and maltose, but TMBP can also bind glucose. The dissociation constant of the TMBP-glucose complex is in the range of 3-8 mM. The binding of glucose does not noticeably change the intrinsic fluorescence of the TMBP. To register protein-glucose binding, we used the fluorescence of the thiol-reactive dye BADAN attached to TMBP. Because the fluorescence of BADAN attached to the cysteine Cys182 of TMBP does not change upon glucose binding, the mutant forms ТМВР/C182S/X_Cys were created. In these mutant proteins, Cys182 is replaced by Ser, removing intrinsic binding site of BADAN and a new dye binding sites were introduced. The largest increase (by 1.4 times) in the intensity of the dye fluorescence was observed upon TMBP/C182S/A14C-BADAN-Glc complex formation. The dissociation constant of this complex is 3.4 ± 0.1 mM. We consider TMBP/C182S/A14C mutant form with attached fluorescent dye BADAN as a good basis for further research aimed to develop of series of TMBP mutant forms with different affinities to glucose labeled with fluorescent dyes.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

PubMed Central

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

Fluorometric graphene oxide-based detection of Salmonella enteritis using a truncated DNA aptamer.

PubMed

Chinnappan, Raja; AlAmer, Saleh; Eissa, Shimaa; Rahamn, Anas Abdel; Abu Salah, Khalid M; Zourob, Mohammed

2017-12-18

The work describes a fluorescence-based study for mapping the highest affinity truncated aptamer from the full length sequence and its integration in a graphene oxide platform for the detection of Salmonella enteriditis. To identify the best truncated sequence, molecular beacons and a displacement assay design are applied. In the fluorescence displacement assay, the truncated aptamer was hybridized with fluorescein and quencher-labeled complementary sequences to form a fluorescence/quencher pair. In the presence of S. enteritidis, the aptamer dissociates from the complementary labeled oligonucleotides and thus, the fluorescein/quencher pair becomes physically separated. This leads to an increase in fluorescence intensity. One of the truncated aptamers identified has a 2-fold lower dissociation constant (3.2 nM) compared to its full length aptamer (6.3 nM). The truncated aptamer selected in this process was used to develop a fluorometric graphene oxide (GO) based assay. If fluorescein-labeled aptamer is adsorbed on GO via π stacking interaction, fluorescence is quenched. However, in the presence of target (S. enteriditis), the labeled aptamers is released from surface to form a stable complex with the bacteria and fluorescence is restored, depending on the quantity of bacteria being present. The resulting assay has an unsurpassed detection limit of 25 cfu·mL -1 in the best case. The cross reactivity to Salmonella typhimurium, Staphylococcus aureus and Escherichia coli is negligible. The assay was applied to analyze doped milk samples for and gave good recovery. Thus, we believe that the truncated aptamer/graphene oxide platform is a potential tool for the detection of S. Enteritidis. Graphical abstract Fluorescently labelled aptamer against Salmonella enteritidis was adsorbed on the surface of graphene oxide by π-stacking interaction. This results in quenching of the fluorescence of the label. Addition of Salmonella enteritidis restores fluorescence, and this effect is used for quantification of this food-borne pathogen.

Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Qiu-Yue; Huang, Wei; Jiang, Xing-Lin; Kang, Yan-Jun

2018-01-01

In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Stöber method. Streptavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cell-probe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system.

Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles

NASA Astrophysics Data System (ADS)

Yang, Boqian; He, Tao; Grauffel, Cédric; Reuter, Nathalie; Roberts, Mary; Gershenson, Anne

2013-03-01

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that Bacillus thuringiensis PI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. This work was supported by the National Institute of General Medical Science of the National Institutes of Health (R01GM060418).

Fluorescent and Magnetic Mesoporous Hybrid Material: A Chemical and Biological Nanosensor for Hg2+ Ions

PubMed Central

Suresh, Moorthy; Anand, Chokkalingam; Frith, Jessica E.; Dhawale, Dattatray S.; Subramaniam, Vishnu P.; Strounina, Ekaterina; Sathish, Clastinrusselraj I.; Yamaura, Kazunari; Cooper-White, Justin J.; Vinu, Ajayan

2016-01-01

We introduce “sense, track and separate” approach for the removal of Hg2+ ion from aqueous media using highly ordered and magnetic mesoporous ferrosilicate nanocages functionalised with rhodamine fluorophore derivative. These functionalised materials offer both fluorescent and magnetic properties in a single system which help not only to selectively sense the Hg2+ ions with a high precision but also adsorb and separate a significant amount of Hg2+ ion in aqueous media. We demonstrate that the magnetic affinity of these materials, generated from the ultrafine γ-Fe2O3 nanoparticles present inside the nanochannels of the support, can efficiently be used as a fluorescent tag to sense the Hg2+ ions present in NIH3T3 fibroblasts live cells and to track the movement of the cells by external magnetic field monitored using confocal fluorescence microscopy. This simple approach of introducing multiple functions in the magnetic mesoporous materials raise the prospect of creating new advanced functional materials by fusing organic, inorganic and biomolecules to create advanced hybrid nanoporous materials which have a potential use not only for sensing and the separation of toxic metal ions but also for cell tracking in bio-separation and the drug delivery. PMID:26911660

Fluorescence imaging and dynamics of intracellular ionic concentrations in single living cells: application to pHi and Mgi variations

NASA Astrophysics Data System (ADS)

Viallet, Pierre M.; Yassine, Mohamed; Salmon, Jean-Marie; Vigo, Jean

1996-05-01

The intracellular concentration of ions such as H+, Hg2+, Ca2+ is known to monitor the activity of many intracellular enzymes. Furthermore these ions are considered as intracellular messengers involved in signal transducing. Moreover recent technological progresses gave rise to the feeling that accurate data are instantly accessible on microvolumes. So the determination of ionic intracellular concentrations has been achieved using fluorescent specific probes and different equipments (Microspectrofluorometer, Flow Cytometer, Numerical Image Analyzer with or without Confocal system), without taking care of the physico-chemical properties of the probe. Unfortunately fluorescent probes are supposed to fill up conflicting requirements in terms of ionic affinity, specificity, fluorescence quantum yield of the free and ion-bound probe, absence of fading and diffusibility out of the cell. Because most of the probes are not so specific than it is claimed, unexpected interactions may obscure the interpretation of results and even make it difficult to get an intracellular calibration curve. Such a situation generally precludes the use of the popular simplest methods of data acquisition and treatment. The scope of this presentation is to point out some underestimated difficulties, to discuss different ways for bypassing some of them and to rationale the use of Videomicrofluorometry.

Quinoline Fluorescent Probes for Zinc - from Diagnostic to Therapeutic Molecules in Treating Neurodegenerative Diseases.

PubMed

Czaplinska, Barbara; Spaczynska, Ewelina; Musiol, Robert

2018-01-01

Fluorescent compounds had gained strong attention due to their wide and appealing applications. Microscopic techniques and visualization are good examples among others. Introduction of fluorescent dyes into microbiology opens the possibility to observe tissues, organisms or organelle with exceptional sensitivity and resolution. Probes for detection of biologically relevant metals as zinc, iron or copper seems to be particularly important for drug design and pharmaceutical sciences. Quinoline derivatives are well known for their good metal affinity and wide spectrum of biological activity. In this regard, molecular sensors built on this scaffold may be useful not only as analytical but also as therapeutic agents. In the present review, application of quinoline moiety in designing of novel fluorescent probes for zinc is presented and discussed. Zinc cations are relevant for vast majority of processes and recently attract a great deal of attention for their role in neurodegenerative diseases. Compounds interacting with Zn2+ may be used for early diagnosis of such disorders, for example the Alzheimer disease. Quinoline-based zinc probes may exert some beneficial role in organism acting as theranostic agents. First preliminary drugs for Alzheimer therapy that are based on quinoline moiety are good example of this trend. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

PubMed Central

Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

2012-01-01

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes.

PubMed

Pélissier, Hélène C; Peters, Winfried S; Collier, Ray; van Bel, Aart J E; Knoblauch, Michael

2008-11-01

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.

GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

PubMed Central

Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

2008-01-01

Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195