scholarly journals Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes.

1985 ◽  
Vol 100 (1) ◽  
pp. 292-296 ◽  
Author(s):  
N McKenna ◽  
J B Meigs ◽  
Y L Wang
Keyword(s):  
Rhodamine B ◽  
Binding Sites ◽  
Cardiac Fibroblasts ◽  
Sulfonyl Chloride ◽  
Fluorescein Isothiocyanate ◽  
Actin Binding ◽  
Experimental Conditions ◽  
Leg Muscles ◽  
Nonmuscle Cells ◽  
Preferential Binding

We have investigated whether living muscle and nonmuscle cells can discriminate between microinjected muscle and nonmuscle actins. Muscle actin purified from rabbit back and leg muscles and labeled with fluorescein isothiocyanate, and nonmuscle actin purified from lamb brain and labeled with lissamine rhodamine B sulfonyl chloride, were co-injected into chick embryonic cardiac myocytes and fibroblasts. When fluorescence images of the two actins were compared using filter sets selective for either fluorescein isothiocyanate or lissamine rhodamine B sulfonyl chloride, essentially identical patterns of distribution were detected in both muscle and nonmuscle cells. In particular, we found no structure that, at this level of resolution, shows preferential binding of muscle or nonmuscle actin. In fibroblasts, both actins are associated primarily with stress fibers and ruffles. In myocytes, both actins are localized in sarcomeres. In addition, the distribution of structures containing microinjected actins is similar to that of structure containing endogenous F-actin, as revealed by staining with fluorescent phalloidin or phallacidin. Our results suggest that, at least under these experimental conditions, actin-binding sites in muscle and nonmuscle cells do not discriminate among different forms of actins.

Structural evidence for asymmetric ligand binding to transthyretin

2015 ◽  
Vol 71 (8) ◽  
pp. 1582-1592 ◽  
Author(s):  
Michele Cianci ◽  
Claudia Folli ◽  
Francesco Zonta ◽  
Paola Florio ◽  
Rodolfo Berni ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Assays ◽  
Experimental Conditions ◽  
Affinity Ligand ◽  
Competition Binding ◽  
Preferential Binding ◽  
Minor Site ◽  
Dynamics Simulations ◽  
Effective Drugs

Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization.

scholarly journals Removal of Rhodamine B from Water Using a Solvent Impregnated Polymeric Dowex 5WX8 Resin: Statistical Optimization and Batch Adsorption Studies

Polymers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 500 ◽  
Author(s):  
Moonis Ali Khan ◽  
Momina ◽  
Masoom Raza Siddiqui ◽  
Marta Otero ◽  
Shareefa Ahmed Alshareef ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rhodamine B ◽  
Binding Sites ◽  
Contact Time ◽  
Adsorption Equilibrium ◽  
Batch Adsorption ◽  
Experimental Conditions ◽  
Impregnated Resin ◽  
Before And After ◽  
Scanning Electron

Herein, commercially available Dowex 5WX8, a cation exchange polymeric resin, was modified through solvent impregnation with t-butyl phosphate (TBP) to produce a solvent impregnated resin (SIR), which was tested for the removal of rhodamine B (RhB) from water in batch adsorption experiments. The effect of SIR dosage, contact time, and pH on RhB adsorption was studied and optimized by response surface methodology (RSM), interaction, Pareto, and surface plots. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were respectively used for characterizing SIR surface morphology and identifying active binding sites before and after RhB adsorption. SEM showed that the pristine SIR surface was covered with irregular size and shape spots with some pores, while RhB saturated SIR surface was non-porous. FTIR revealed the involvement of electrostatic and π–π interactions during RhB adsorption on SIR. Dosage of SIR, contact time, and their interaction significantly affected RhB adsorption on SIR, while pH and its interaction with dosage and contact time did not. The optimum identified experimental conditions were 0.16 g of SIR dose and 27.66 min of contact time, which allowed for 98.45% color removal. Moreover, RhB adsorption equilibrium results fitted the Langmuir isotherm with a maximum monolayer capacity (qmax) of 43.47 mg/g.

scholarly journals Perturbations of Drosophila alpha-actinin cause muscle paralysis, weakness, and atrophy but do not confer obvious nonmuscle phenotypes.

1992 ◽  
Vol 116 (4) ◽  
pp. 911-922 ◽  
Author(s):  
E M Roulier ◽  
C Fyrberg ◽  
E Fyrberg
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Single Gene ◽  
Actin Binding ◽  
Muscle Paralysis ◽  
Leg Muscles ◽  
Nonmuscle Cell ◽  
Actin Binding Domain ◽  
Nonmuscle Cells ◽  
Cross Linker

We have investigated accumulation of alpha-actinin, the principal cross-linker of actin filaments, in four Drosophila fliA mutants. A single gene is variably spliced to generate one nonmuscle and two muscle isoforms whose primary sequence differences are confined to a peptide spanning the actin binding domain and first central repeat. In fliA3 the synthesis of an adult muscle-specific isoform is blocked in flight and leg muscles, while in fliA4 the synthesis of nonmuscle and both muscle-specific isoforms is severely reduced. Affected muscles are weak or paralyzed, and, in the case of fliA3, atrophic. Their myofibrils, while structurally irregular, are remarkably normal considering that they are nearly devoid of a major contractile protein. Also surprising is that no obvious nonmuscle cell abnormalities can be discerned despite the fact that both the fliA1- and fliA4-associated mutations perturb the nonmuscle isoform. Our observations suggest that alpha-actinin stabilizes and anchors thin filament arrays, rather than orchestrating their assembly, and further imply that alpha-actinin function is redundant in both muscle and nonmuscle cells.

scholarly journals Silver Nanoparticles Functionalized by Fluorescein Isothiocyanate or Rhodamine B Isothiocyanate: Fluorescent and Plasmonic Materials

2021 ◽  
Vol 11 (6) ◽  
pp. 2472
Author(s):  
Ilaria Fratoddi ◽  
Chiara Battocchio ◽  
Giovanna Iucci ◽  
Daniele Catone ◽  
Antonella Cartoni ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Rhodamine B ◽  
Photoelectron Spectroscopy ◽  
Transient Absorption ◽  
Fluorescein Isothiocyanate ◽  
Dye Molecules ◽  
Chemical Surface ◽  
Vis Spectroscopy ◽  
Rhodamine B Isothiocyanate ◽  
Fluorescent Molecules

This paper presents the synthesis of silver nanoparticles (AgNPs) functionalized with fluorescent molecules, in particular with xanthene-based dyes, i.e., fluorescein isothiocyanate (FITC, λmax = 485 nm) and rhodamine B isothiocyanate (RITC, λmax = 555 nm). An in-depth characterization of the particle–dye systems, i.e., AgNPs–RITC and AgNPs–FITC, is presented to evaluate their chemical structure and optical properties due to the interaction between their plasmonic and absorption properties. UV–Vis spectroscopy and the dynamic light scattering (DLS) measurements confirmed the nanosize of the AgNPs–RITC and AgNPs–FITC. Synchrotron radiation X-ray photoelectron spectroscopy (SR-XPS) was used to study the chemical surface functionalization by structural characterization, confirming/examining the isothiocyanate–metal interaction. For AgNPs–RITC, in which the plasmonic and fluorescence peak are not superimposed, the transient dynamics of the dye fluorescence were also studied. Transient absorption measurements showed that by exciting the AgNPs–RITC sample at a wavelength corresponding to the AgNP plasmon resonance, it was possible to preferentially excite the RITC dye molecules attached to the surface of the NPs with respect to the free dye molecules in the solution. These results demonstrate how, by combining plasmonics and fluorescence, these AgNPs can be used as promising systems in biosensing and imaging applications.

scholarly journals The Efficient Photocatalytic Degradation of Organic Pollutants on the MnFe2O4/BGA Composite under Visible Light

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1276
Author(s):  
Qian Li ◽  
Xiaoyu Jiang ◽  
Yongfu Lian
Keyword(s):  
Rhodamine B ◽  
Synergetic Effect ◽  
Reaction Rate Constant ◽  
Experimental Conditions ◽  
First Order Kinetics ◽  
Boron Doped ◽  
Fenton Catalyst ◽  
Aerogel Composite ◽  
Doped Graphene ◽  
Apparent Reaction Rate Constant

The MnFe2O4/BGA (boron-doped graphene aerogel) composite was prepared by hydrothermal treatment of MnFe2O4 particles, boric acid, and graphene oxide. When applied as a photo-Fenton catalyst for the degradation of rhodamine B, the MnFe2O4/BGA composite yielded a degradation efficiency much higher than the sum of those of individual MnFe2O4 and BGA under identical experimental conditions, indicating a strong synergetic effect established between MnFe2O4 and BGA. The catalytic degradation of rhodamine B was proved to follow pseudo first-order kinetics, and the apparent reaction rate constant on the MnFe2O4/BGA composite was calculated to be three- and seven-fold that on BGA and MnFe2O4, respectively. Moreover, the MnFe2O4/BGA composite also demonstrated good reusability and could be reused for four cycles without obvious loss of photocatalytic activity.

scholarly journals Effects of diadenosine polyphosphates and seminal fluid vesicles on rabbit sperm cells

Reproduction ◽  
2003 ◽  
pp. 827-835 ◽  
Author(s):  
A Minelli ◽  
L Liguori ◽  
I Bellezza ◽  
T Renieri ◽  
C Castellini
Keyword(s):  
High Performance ◽  
Acrosome Reaction ◽  
Seminal Fluid ◽  
Membrane Vesicles ◽  
Fluorescein Isothiocyanate ◽  
Plasma Electron ◽  
Sperm Cells ◽  
Experimental Conditions ◽  
Diadenosine Polyphosphates ◽  
Rabbit Spermatozoa

Membrane vesicles were isolated from rabbit seminal plasma. Electron microscopy analyses showed the presence of numerous small, round vesicles with a diameter of about 70 nm. Determination of enzyme activities was carried out by high performance liquid chromatography and showed that the vesicles can degrade the diadenosine polyphosphates (ApnA), Ap3A and Ap4A and ATP and ADP, but not AMP. Studies of the degradation of diadenosine compounds by the vesicles present in seminal fluid showed an increasing production of AMP as the by-product and a time-dependent generation of dephosphorylated products consistent with the presence of ecto-ATP diphosphophosphatase (ecto-apyrase). In the presence of rabbit spermatozoa, AMP did not accumulate because 5'nucleotidase and adenosine deaminase, present at the surface of sperm cells, transformed AMP into adenosine and inosine. The effects of seminal fluid vesicles and diadenosine compounds on the acquisition of fertilizing capacity by rabbit spermatozoa were evaluated by Pisum sativum agglutinin fluorescein isothiocyanate conjugated staining. The results obtained with uncapacitated spermatozoa showed that the capacitating effector BSA could be substituted efficiently by the addition of diadenosine compounds and vesicles previously incubated for 2 h to the capacitative medium. Under these experimental conditions, the spontaneous acrosome reaction rate was not increased. Capacitated rabbit spermatozoa did not undergo acrosome reaction when l-alpha-lysophosphatidylcholine was substituted by diadenosine compounds previously incubated with vesicles. In conclusion, this study has shown that rabbit seminal fluid vesicles can degrade diadenosine compounds to AMP and that the addition of the vesicles and diadenosine compounds to uncapacitated rabbit spermatozoa favours the acquisition of the fertilizing capacity.

scholarly journals Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

1994 ◽  
Vol 126 (2) ◽  
pp. 433-443 ◽  
Author(s):  
A McGough ◽  
M Way ◽  
D DeRosier
Keyword(s):  
Image Analysis ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Outer Face ◽  
Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

scholarly journals Tissue-Restricted Expression of the Cardiac α-Myosin Heavy Chain Gene Is Controlled by a Downstream Repressor Element Containing a Palindrome of Two Ets-Binding Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7243-7258 ◽  
Author(s):  
Madhu Gupta ◽  
Radovan Zak ◽  
Towia A. Libermann ◽  
Mahesh P. Gupta
Keyword(s):  
Gene Regulation ◽  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Binding Sites ◽  
Cardiac Myocyte ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Nonmuscle Cells ◽  
Muscle Gene

ABSTRACT The expression of the α-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in α-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac α-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the α-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the α-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the α-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the α-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the α-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the α-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the α-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

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