RESUMEN
BACKGROUND: Flagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical '9 + 2' cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain. RESULTS: We found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus. CONCLUSION: It appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.
Asunto(s)
Chlamydomonas/metabolismo , Flagelos/metabolismo , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Filogenia , Proteínas de Plantas/química , Dominios Proteicos , Transporte de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo. Another significant factor which is overlooked is the effects of macromolecular crowding and its consequences in the actual biochemical and physiological environment. Such influences of crowding, its modification and physiological parameters have been reported. The present study investigates the effect of molecular crowding on binding characteristics of Salmon sperm DNA with Bovine hemoglobin and their reconstitutes in presence of molecular crowders viz., Poly ethylene glycol (PEG) and Dextran of different molecular weight by fluorescence, UV visible spectroscopic technique at different temperatures. The results showed that BHb fluorescence was quenched by sDNA through static quenching mechanism which is enhanced in presence of polymers. The number of binding sites 'n' and binding constants 'K' were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely ∆H°, ∆G°, T∆S° were studied at different temperatures and the results indicate that hydrophobic forces are predominant in the sDNA-BHb complex. Negative ∆G° values imply that the binding process is spontaneous.
Asunto(s)
ADN/química , Dextranos/química , Hemoglobinas/química , Polietilenglicoles/química , Termodinámica , Animales , Bovinos , Sustancias Macromoleculares/química , Masculino , Salmón , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , EspermatozoidesRESUMEN
Biological macromolecules evolve and function within intracellular environments that are crowded with other macromolecules. Crowding results in surprisingly large quantitative effects on both the rates and the equilibria of interactions involving macromolecules, but such interactions are commonly studied outside the cell in uncrowded buffers. The addition of high concentrations of natural and synthetic macromolecules to such buffers enables crowding to be mimicked in vitro, and should be encouraged as a routine variable to study. In this study, we propose to understand the changes in DNA character and its modulation in presence of macromolecules such as PEG with reference to binding parameters to amino acids using fluorescence enhancement.
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Aminoácidos/química , ADN/química , Aminoácidos/análisis , ADN/análisis , Fluorescencia , Sustancias Macromoleculares/análisis , Sustancias Macromoleculares/química , Polietilenglicoles/química , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
The protein-nanoparticle interface plays a crucial role in drug binding and stability, in turn enhancing efficacy in targeted drug delivery. In the present study, whey protein ß-lactoglobulin (BLG) is conjugated with gold nanoparticles (AuNP) and its interaction with curcumin (CUR) and gemcitabine (GEM) has been explored. Further, AuNP-BLG conjugate interactions with anticancer drugs were characterized using dynamic light scattering (DLS), zeta potential, UV-visible, Raman spectroscopy, fluorescence, circular dichroism along with molecular dynamics simulation. The cytotoxicity studies were performed using breast cancer cell lines (MCF-7). â¼8 µM of BLG resides on AuNP (â¼29 nm) surface revealed by DLS. Raman scattering of AuNP-BLG conjugate showed orientation of the central calyx of BLG towards solvent. BLG fluorescence confirmed the interaction between AuNP-BLG conjugate with drugs and indicated strong binding and affinity (for CUR KD = 3.71 x 108 M -1, n = 1.83, and for GEM KD = 3.78 x 103 M -1, n = 0.94), enhanced in the presence of AuNP. CD and Raman analysis exhibited selective hydrophilic and hydrophobic conformations induced by drug binding. Computational studies on BLG-drug complexes revealed that the residues Pro38, Leu39 and Met107 are largely associated with CUR binding, while GEM interaction is via hydrophilic contacts which significantly matches with spectroscopic investigation. IC50 values were calculated for all components of this loading system on MCF-7. The possible mechanisms of interaction between AuNP-BLG with anticancer drugs has been explored at the molecular level. We believe that these conjugates could be considered in the targeted drug delivery studies for cancer research.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Antineoplásicos , Curcumina , Nanopartículas del Metal , Antineoplásicos/farmacología , Dicroismo Circular , Curcumina/química , Oro/química , Lactoglobulinas/química , Nanopartículas del Metal/químicaRESUMEN
Physical and dosimetric characteristics of HDMLC were studied for SRS6, 6, and 10 MV X-rays from Novalis Tx. This in-built tertiary collimator consists of 60 pairs (32 × 0.25 cm; 26 × 0.5 cm and 2 × 0.7 cm) of leaves. Properties of HDMLC studied included alignment, readout and radiation field congruence, radiation penumbra, accuracy and reproducibility of leaf position and gap width, static and dynamic leaf shift, tongue-and-groove effect, leaf transmission and leakage, leaf travel speed, and delivery of dynamic conformal arc and IMRT. All tests were performed using a calibrated ionization chamber, film dosimetry and DynaLog file analysis. Alignment of leaves with isocenter plane was better than 0.03 cm at all gantry and collimator positions. The congruence of HDMLC readout and radiation field agreed to within ± 0.03 cm for filed sizes ranging from 1 × 1 to 20 × 20 cm2. Mean 80% to 20% penumbra width parallel (perpendicular) to leaf motion was 0.24 ± 0.05 (0.21 ± 0.02) cm, 0.37 ± 0.12 (0.29 ± 0.07) cm, and 0.51 ± 0.13 (0.43± 0.07) cm for SRS6, 6, and 10 MV X-rays, respectively. Circular field penumbra was comparable to corresponding square field. Average penumbra of 1 × 20 cm2 field was effectively constant over off-axis positions of up to 12 cm with mean value of 0.16 (± 0.01) cm at 1.5 cm depth and 0.38 (± 0.04) cm at 10 cm depth. Minimum and maximum effective penumbra along the straight diagonal edge of irregular fields increased from 0.3 and 0.32 cm at 70° steep angle to 0.35 and 0.56 cm at 20° steep angle. Modified Picket Fence test showed average FWHM of 0.18 cm and peak-to-peak distance of 1.99 cm for 0.1 cm band and 2 cm interband separation. Dynamic multileaf collimation (DMLC) output factor remained within ± 1% for 6 MV and ± 0.5% for 10 MV X-rays at all gantry positions, and was reproducible within ± 0.5% over a period of 14 months. The static leaf shift was 0.03 cm for all energies, while dynamic leaf shift was 0.044 cm for 10 MV and 0.039 cm for both SRS6 and 6 MV X-rays. The dose depression and corresponding tongue-and-groove size were 24% and 0.17 cm for 6 MV and 19% and 0.20 cm for 10 MV X-rays. Average transmission through HDMLC was 1.09%, 1.14% and 1.34% for SRS6, 6 and 10 MV X-rays. Analysis of DynaLog files for leaf speed test in arc dynamic mode, delivery test of dynamic conformal arc, and step-and-shoot and sliding window IMRT showed at least 95% or more of the error counts had misplacements < 0.2 cm, with maximum root mean square (RMS) error value calculated at 0.13cm. Accurate and reproducible leaf position and gap width, and less leakage and small consistent penumbra over the fields demonstrate HDMLC suitable for high-dose resolution SRS and IMRT.
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Planificación de la Radioterapia Asistida por Computador/métodos , Radioterapia de Intensidad Modulada/métodos , Humanos , Aceleradores de Partículas/instrumentación , Garantía de la Calidad de Atención de Salud/normas , Radiometría/instrumentación , Dosificación Radioterapéutica , Planificación de la Radioterapia Asistida por Computador/instrumentación , Planificación de la Radioterapia Asistida por Computador/normas , Radioterapia de Intensidad Modulada/instrumentación , Radioterapia de Intensidad Modulada/normasRESUMEN
Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules, such as, aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T-Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.
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Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Heparina/análisis , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodos , Trombina/análisis , Aniones/química , Benzotiazoles/análisis , Benzotiazoles/química , Cationes/química , Dicroismo Circular , Colorantes Fluorescentes , Humanos , EspectrofotometríaRESUMEN
Protamine, a polycation, is biologically and medically relevant protein. Protamine exhibits a wide array of functions in biological processes like gene transfer, tissue and organogenesis, cell reproduction, etc. Medically, Protamine is the only clinically approved antidote for Heparin and is routinely used in various surgical interventions, and hence controlling Protamine dosing in patients is very crucial. Taking into account the medical significance of Protamine, designing simple, reliable and sensitive fluorescence sensors is highly desirable. In this work, we propose one such sensitive and reliable fluorescent sensor which is based on a template of dye-polyelectrolyte assembly constituting a molecular rotor dye, Thioflavin-T and an anionic synthetic polyelectrolyte, polystyrene sulfonate. The addition of Protamine, prompts drastic modulations in spectral features of dye-polyelectrolyte assembly which enables sensitive detection of Protamine in aqueous solution. Apart from sensitive detection, our sensing platform aids in highly selective sensing of Protamine compared to other proteins. Moreover, our sensor system is constructed on label-free, inexpensive, commercially available molecules posing as an advantage over other sensor systems which involve laborious synthesis protocols. Most importantly, our sensor template is able to sense Protamine in diluted serum sample, indicating the potential practical utility of our sensor system.
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Aniones , Benzotiazoles/química , Polielectrolitos/química , Protaminas/química , Animales , Colorantes/química , Electrólitos , Fluorescencia , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Fotoquímica , Poliestirenos/química , Albúmina Sérica Humana/química , Espectrometría de Fluorescencia , TemperaturaRESUMEN
This investigation understands the interaction between lyophilized crude Viper snake venom (Doboia russellie) and Silver nanoparticles (SNPs) using biophysical and biochemical approaches. SNPs were synthesized by chemical reduction method and characterized using UV-Visible spectroscopy, Dynamic Light Scattering (DLS) and Transmission electron microscope (TEM). The average hydrodynamic size of SNPs was found to be 52 nm with 0.261 PDI. TEM image revealed the spherical shape of SNP. Interaction of SNPs and viper venom was resulted in the formation of complex which was confirmed by using DLS technique. Spectroscopic results showed an increase in absorbance intensity of venom upon interaction with SNPs which indicated interaction with venom proteins. Fluorescence spectroscopic data revealed the quenching in the fluorescence intensity of viper venom upon incubation with varying concentration of SNPs. The results obtained by biochemical assays (Protease and whole blood clotting test) revealed the inhibition of venom action due to presence of silver nanoparticles. The activity of protease enzyme was found to be decreased (10-13% reduction) in presence of silver nanoparticles. Prolonged clotting time (two fold) of viper venom upon interaction with SNPs compared to native crude viper venom was observed. The overall results confirmed the inhibition action of silver nanoparticles against viper venom.
RESUMEN
Nanomaterials have been used widely for delivery of therapeutic agents. Protein-nanoparticle (NP) complexes have gained importance as vehicles for targeted drug delivery due to increased ease of administration, stability and half-life of drug, and reduced toxic side effects. Designing of phospholipid-bovine serum albumin (BSA) complexes and stealth NPs with BSA has paved the way for drug delivery carriers with prolonged blood circulation times. Preformed albumin corona has shown to decrease non-specific association and thereby reduce the clearance rate. Albumin corona has enabled the localization of drug carriers in specific tissues such as liver and heart, thus regulating biodistribution. Tailored albumin-NP conjugates have also enabled controlled degradation of NP and drug release. However, the binding of albumin with NP is associated with conformational and functional modulations in protein as observed with silver, gold and superparamagnetic iron oxide NPs. In this review, we highlight the various potential albumin-NP hybrids as nano drug carriers.