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Süleyman Cinar, Claus Czeslik
We compare the binding of an inhibitor with that of a natural peptide to Ca2+ saturated calmodulin (holo-CaM). As inhibitor we have chosen trifluoperazine (TFP) that is inducing a huge conformational change of holo-CaM from the open dumbbell-shaped to the closed globular conformation upon binding. On the other hand, melittin is used as model peptide, which is a well-known natural binding partner of holo-CaM. The experiments are carried out as a function of pressure to reveal the contribution of volume or packing effects to the stability of the calmodulin-ligand complexes...
March 16, 2018: Biochimica et Biophysica Acta
Alberto Bresciani, Maria Carolina Spiezia, Roberto Boggio, Cristina Cariulo, Anja Nordheim, Roberta Altobelli, Kirsten Kuhlbrodt, Celia Dominguez, Ignacio Munoz-Sanjuan, John Wityak, Valentina Fodale, Deanna M Marchionini, Andreas Weiss
Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions...
2018: PloS One
Rachel Coleman, Darci J Trader
To attenuate an over-abundance of cellular protein, it has been hypothesized that the 20S core particle (20S CP) of the proteasome can be chemically stimulated to degrade proteins into non-toxic peptides more quickly. Screening for small molecule 20S CP stimulators is typically performed with a reporter peptide composed of four amino acids and a coumarin group that is released upon proteasome-mediated hydrolysis to generate a fluorescent signal. Screening with this small reporter can lead to false negatives because the reporter peptide is rapidly turned-over without stimulation...
March 19, 2018: ACS Combinatorial Science
Riti Sharan, Hee-Jeong Yang, Preeti Sule, Jeffrey D Cirillo
Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained...
February 26, 2018: Journal of Visualized Experiments: JoVE
Gembu Maryu, Haruko Miura, Yoichi Uda, Akira T Komatsubara, Michiyuki Matsuda, Kazuhiro Aoki
Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions...
March 17, 2018: Cell Structure and Function
Jared T Hammill, Daniel C Scott, Jaeki Min, Michele C Connelly, Gloria Holbrook, Fangyi Zhu, Amy Matheny, Lei Yang, Bhuvanesh Singh, Brenda A Schulman, R Kiplin Guy
We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit...
March 16, 2018: Journal of Medicinal Chemistry
A Susanne Dittrich, Iris Kühbandner, Stefanie Gehrig, Verena Rickert-Zacharias, Matthew Twigg, Sabine Wege, Clifford C Taggart, Felix Herth, Carsten Schultz, Marcus A Mall
Neutrophil elastase (NE) is a key risk factor for severity of cystic fibrosis (CF) lung disease. Recent studies identified increased NE activity on the surface of airway neutrophils from CF-like mice and patients with CF. However, the role of surface-bound NE in CF lung disease remains unknown. We, therefore, determined the relationship between surface-bound NE activity and severity of lung disease in CF.Surface-bound NE activity was measured on sputum neutrophils from 35 CF patients and 8 healthy controls using novel lipidated Foerster resonance energy transfer (FRET) reporters and correlated with free NE activity, neutrophil counts, IL-8, myeloperoxidase and antiproteases in sputum supernatant, and with lung function parameters...
March 15, 2018: European Respiratory Journal: Official Journal of the European Society for Clinical Respiratory Physiology
Magdalena Kolasa, Joanna Solich, Agata Faron-Górecka, Dariusz Żurawek, Paulina Pabian, Sylwia Łukasiewicz, Maciej Kuśmider, Kinga Szafran-Pilch, Marta Szlachta, Marta Dziedzicka-Wasylewska
Recently, it has been shown that serotonin 5-HT1A receptor interacts with dopamine D2 receptor in vitro. However, the existence of 5-HT1A -D2 heteromers in native tissue remains unexplored. In the present study, we investigated 5-HT1A -D2 receptor heteromerization in mice treated acutely or chronically with paroxetine (10 mg/kg) or risperidone (0.05 mg/kg). Receptor heteromerization was visualized and quantified in the mouse brain by in situ proximity ligation assay (PLA). Additionally, we aimed to determine the cellular localization of 5-HT1A -D2 receptor heteromers in mouse adult primary neuronal cells by immunofluorescent staining with markers for astrocytes (GFAP) and neurons (NeuN and MAP2)...
March 12, 2018: Neuroscience
Niels Vandenberk, Anders Barth, Doortje Borrenberghs, Johan Hofkens, Jelle Hendrix
Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces and structure. Single-molecule (sm) FRET additionally allows real-time quantifications of conformation and conformational dynamics. SmFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing dsDNA molecules...
March 15, 2018: Journal of Physical Chemistry. B
Giacomo Reina, Ngoc Do Quyen Chau, Yuta Nishina, Alberto Bianco
Several studies have demonstrated the ability of graphene oxide (GO) to efficiently adsorb small-interfering RNA (siRNA) on its surface and to transport it into cells. However, studies on whether and how siRNA interacts with GO are still inconclusive. In this context, understanding the interaction between GO and siRNA is fundamental to design new efficient gene silencing tools. In this work, the interactions between GO and siRNA molecules were systematically investigated. We focused on how the GO size, oxygenated groups present on the surface and chemical functionalization affect the double helix siRNA structure, using gel electrophoresis, UV-Vis spectroscopy, fluorescence resonance energy transfer (FRET) and circular dichroism (CD)...
March 15, 2018: Nanoscale
Germano Orrù, Mauro Giovanni Carta, Alessia Bramanti
Background: Several studies have shown that the Single Nucleotide Polymorphism (SNP) in the CACAN1C gene, rs1006737, is related to different mood disorder illnesses, such as bipolar disorder and schizophrenia. Current day molecular procedures for allele detection of this gene can be very expensive and time consuming. Hence, a sensitive and specific molecular procedure for detecting these mutations in a large number of subjects is desirable, especially for research groups who have no complex laboratory equipment...
2018: Clinical Practice and Epidemiology in Mental Health: CP & EMH
Sascha Suffner, Nadine Gerstenberg, Maria Patra, Paula Ruibal, Ahmed Orabi, Michael Schindler, Volker Bruss
During hepatitis B virus infections subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (CL) and between TM2 and HCR (LL) are located in the cytosol and in the ER lumen, respectively. To define domains of S mediating oligomerization during SVP morphogenesis S mutants were characterized by expression in transiently transfected cells...
March 14, 2018: Journal of Virology
Laurel M Oldach, Kirill Gorshkov, William T Mills, Jin Zhang, Mollie K Meffert
Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the post-transcriptional level, and can undergo mitogen-activated protein kinase (MAPK)-mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR-RNA-binding protein (TRBP). However, molecular and spatio-temporal details of this critical control mechanism remained unknown...
March 14, 2018: Molecular Biology of the Cell
Daniel Alejandro Peñalva, Silvia Antollini, Ernesto Ambroggio, Marta I Aveldaño, Maria Laura Fanani
In rat sperm head, sphingomyelin (SM) species that contain very long chain PUFA (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalysed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (LUVs, GUVs) containing phosphatidylcholine and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated, and more rapidly, than P-Cer...
March 15, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Audrey Kelner, Nuno Leitão, Mireille Chabaud, Myriam Charpentier, Fernanda de Carvalho-Niebel
Spatiotemporal changes in cellular calcium (Ca2+ ) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments...
2018: Frontiers in Plant Science
Claudia Conte, Francesca Mastrotto, Vincenzo Taresco, Aleksandra Tchoryk, Fabiana Quaglia, Snjezana Stolnik, Cameron Alexander
In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG (polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhance cell internalization of the particles and their drug cargo...
March 10, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
Chantal C Clark, Mirjam M Mebius, Steven de Maat, Aloysius G M Tielens, Philip G de Groot, Rolf T Urbanus, Rob Fijnheer, Bouke P C Hazenberg, Jaap J van Hellemond, Coen Maas
ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system...
March 2018: Thrombosis and Haemostasis
Katharina Schleicher, Manuela Zaccolo
3',5'-cyclic adenosine monophosphate (cAMP) signalling plays a major role in the cardiac myocyte response to extracellular stimulation by hormones and neurotransmitters. In recent years, evidence has accumulated demonstrating that the cAMP response to different extracellular agonists is not uniform: depending on the stimulus, cAMP signals of different amplitudes and kinetics are generated in different subcellular compartments, eliciting defined physiological effects. In this review, we focus on how real-time imaging using fluorescence resonance energy transfer (FRET)-based reporters has provided mechanistic insight into the compartmentalisation of the cAMP signalling pathway and allowed for the precise definition of the regulation and function of subcellular cAMP nanodomains...
March 13, 2018: Journal of Cardiovascular Development and Disease
Laxman S Walekar, Peidong Hu, Hamed Vafaei Molamahmood, Mingce Long
The integrated system of pyrene and cetyltrimethyl ammonium bromide (CTAB) capped silver nanoparticles (AgNPs) with a distance (r) of 2.78nm has been developed for the detection of Hg (II) and pyrene dimer. The interaction between pyrene and AgNPs results in the fluorescence quenching of pyrene due to the energy transfer, whose mechanism can be attributed to the Forster Resonance Energy Transfer (FRET) supported by experimental observation and theoretical calculations. The developed probe shows a highly selective and sensitive response towards Hg (II) probably due to the amalgam formation, which results in the fluorescence recovery (90%) of pyrene and color change of solution from yellowish brown to colorless...
March 7, 2018: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
Adejoke N Kolawole, Valentine T Akinladejo, Olusola O Elekofehinti, Afolabi C Akinmoladun, Ayodele O Kolawole
Aldehyde dehydrogenases (ALDHs) are a diverse family of enzymes that catalyze the NAD(P)+ -dependent detoxification of toxic aldehyde compounds. ALDHs are also involved in non-enzymatic ligand binding to endobiotics and xenobiotics. Here, the enzyme crucial non-canonical and non-catalytic interaction with kolaflavanone, a component of kolaviron, and a major bioflavonoid isolated from Garcinia kola (Bitter kola) was characterized by various spectroscopic and in silico approaches under stimulated physiological condition...
February 27, 2018: Bioorganic Chemistry
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