Comparative Study of Toluidine Blue O and Methylene Blue Binding to Lysozyme and Their Inhibitory Effects on Protein Aggregation.
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ABSTRACT:
SUBMITTER: Saha B
PROVIDER: S-EPMC6044680 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The aggregates of microtubule-associated protein Tau are considered as a major hallmark of Alzheimer's disease. Tau aggregates accumulate intracellularly leading to neuronal toxicity. Numerous approaches have been targeted against Tau protein aggregation, which include application of synthetic and natural compounds. Toluidine blue is a basic dye of phenothiazine family, which on irradiation with a 630 nm light gets converted into a photoexcited form, leading to generation of singlet oxygen species. Methylene blue is the parent compound of toluidine blue, which has been reported to be potent against tauopathy. In the present work, we studied the potency of toluidine blue and photoexcited toluidine blue against Tau aggregation. Biochemical and biophysical analyses using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ThS fluorescence, circular dichroism spectroscopy, and electron microscopy suggested that toluidine blue inhibited the aggregation of Tau in vitro. The photoexcited toluidine blue potentially dissolved the matured Tau fibrils, which indicated the disaggregation property of toluidine blue. The cell biology studies including the cytotoxicity assay and reactive oxygen species (ROS) production assay suggested toluidine blue to be a biocompatible dye as it reduced ROS levels and cell death. The photoexcited toluidine blue modulates the cytoskeleton network in cells, which was supported by immunofluorescence studies of neuronal cells. The studies in a UAS Tau E14 transgenic Drosophila model suggested that photoexcited toluidine blue was potent to restore the survival and memory deficits of Drosophila. The overall finding of our studies suggested toluidine blue to be a potent molecule in rescuing the Tau-mediated pathology by inhibiting its aggregation, reducing the cell death, and modulating the tubulin levels and behavioral characteristics of Drosophila. Thus, toluidine blue can be addressed as a potent molecule against Alzheimer's disease.
| S-EPMC6854831 | biostudies-literature
Project description:The synthesis of new phenothiazine derivatives, analogs of Methylene Blue, is of particular interest in the design of new drugs, as well as in the development of a new generation of agents for photodynamic therapy. In this study, two new derivatives of phenothiazine, i.e., 3,7-bis(4-aminophenylamino)phenothiazin-5-ium chloride dihydrochloride (PTZ1) and 3,7-bis(4-sulfophenylamino)phenothiazin-5-ium chloride (PTZ2), are synthesized for the first time and characterized by NMR, IR spectroscopy, HRMS and elemental analysis. The interaction of the obtained compounds PTZ1 and PTZ2 with salmon sperm DNA is investigated. It is shown by UV-Vis spectroscopy and DFT calculations that substituents in arylamine fragments play a crucial role in dimer formation and interaction with DNA. In the case of PTZ1, two amine groups promote H-aggregate formation and DNA interactions through groove binding and intercalation. In the case of PTZ2, sulfanilic acid fragments prevent any dimer formation and DNA binding due to electrostatic repulsion. DNA interaction mechanisms are studied and confirmed by UV-vis and fluorescence spectroscopy in comparison with Methylene Blue. The obtained results open significant opportunities for the development of new drugs and photodynamic agents.
| S-EPMC8198855 | biostudies-literature
Project description:Methylene blue (MB) is a synthetic phenothiazine dye that, in the last years, has generated much debate about whether it could be a useful therapeutic drug for tau-related pathologies, such as Alzheimer's disease (AD). However, the molecular mechanism of action is far from clear. Recently we reported that MB activates the plasma membrane Ca2+-ATPase (PMCA) in membranes from human and pig tissues and from cells cultures, and that it could protect against inactivation of PMCA by amyloid β-peptide (Aβ). The purpose of the present study is to further examine whether the MB could also modulate the inhibitory effect of tau, another key molecular marker of AD, on PMCA activity. By using kinetic assays in membranes from several tissues and cell cultures, we found that this phenothiazine was able to block and even to completely reverse the inhibitory effect of tau on PMCA. The results of this work point out that MB could mediate the toxic effect of tau related to the deregulation of calcium homeostasis by blocking the impairment of PMCA activity by tau. We then could conclude that MB could interfere with the toxic effects of tau by restoring the function of PMCA pump as a fine tuner of calcium homeostasis.
| S-EPMC6678728 | biostudies-literature
Project description:Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 ?M, and it had an IC50 of 31 ?M in human ?1?2?2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the ?2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of ?1?2 receptors to MB was similar to that observed in ?1?2?2 receptors, indicating that MB's action via the benzodiazepine or Zn2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of ?1 F64 to A and ?2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.
| S-EPMC5506546 | biostudies-literature
Project description:The accelerating spread of drug-resistant bacteria is creating demand for novel antibiotics. Bactericidal enzymes, such as human lysozyme (hLYZ), are interesting drug candidates due to their inherent catalytic nature and lack of susceptibility to the resistance mechanisms typically directed toward chemotherapeutics. However, natural antibacterial enzymes have their own limitations. For example, hLYZ is susceptible to pathogen derived inhibitory proteins, such as Escherichia coli Ivy. Here, we describe proof of concept studies demonstrating that hLYZ can be effectively redesigned to evade this potent lysozyme inhibitor. Large combinatorial libraries of hLYZ were analyzed using an innovative screening platform based on microbial coculture in hydrogel microdroplets. Isolated hLYZ variants were orders of magnitude less susceptible to E. coli Ivy yet retained high catalytic proficiency and inherent antibacterial activity. Interestingly, the engineered escape variants showed a disadvantageous increase in susceptibility to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein, MliC. Thus, while we have achieved our original objective with respect to escaping E. coli Ivy, engineering hLYZ for broad-spectrum evasion of proteinaceous inhibitors will require consideration of the complex and varied determinants that underlie molecular recognition by these emerging virulence factors.
| S-EPMC4445656 | biostudies-literature
Project description:In this paper, we study the interaction of a small dye molecule, namely, methylene blue (MB) with graphene surfaces using surface plasmon resonance (SPR). We show that by utilizing all of the parameters of the SPR angular dip and exploiting the fact that MB absorbs light at the operating wavelength, it is possible to detect the binding of small molecules that would otherwise not give a significant signal. The binding of MB to unmodified graphene is found to be stronger than that for gold. By studying the interaction at modified surfaces, we demonstrate that electrostatic effects play a dominant role in the binding of MB on to graphene. Furthermore, following the binding kinetics at various concentrations allows us to estimate apparent equilibrium binding and rate constants for the interaction of MB with graphene.
| S-EPMC6644572 | biostudies-literature
Project description:BACKGROUND:Although methylene blue (MB) had long been proposed to counteract the effects of cyanide (CN) intoxication, research on its mechanisms of action and efficacy has been abandoned for decades. Recent studies on the benefits of MB in post-anoxic injuries have prompted us to reexamine the relevance of this historical observation. METHODS:Our study was performed in adult male Sprague-Dawley rats and on HEK293T epithelial cells. First, the effects and toxicity of MB (0-80?mg/kg) on circulation and metabolism were established in four urethane-anesthetized rats. Then nine rats received a lethal infusion of a solution of KCN (0.75?mg/kg/min) and were treated by either saline or MB, at 20?mg/kg, a dose that we found to be innocuous in rat and to correspond to a dose of about 4?mg/kg in humans. MB was also administered 5?min after the end of a sub-lethal exposure to CN in a separate group of 10 rats. In addition, ATP/ADP ratio, ROS production, mitochondrial membrane potential (??m) and cellular O2 consumption rate (OCR) were determined in HEK293T cells exposed to toxic levels of CN (200?µM for 10?min) before and after applying a solution containing MB (1-100?µM for 10?min). RESULTS:Methylene blue was found to be innocuous up to 50?mg/kg. KCN infusion (0.75?mg/kg/min) killed all animals within 7-8?min. MB (20?mg/kg) administered at the same time restored blood pressure, cardiac contractility and limited O2 deficit, allowing all the animals to survive, without any significant methemoglobinemia. When administered 5?min after a non-lethal CN intoxication, MB sped up the recovery of lactate and O2 deficit. Finally, MB was able to decrease the production of ROS and restore the ATP/ADP ratio, ??m as well as OCR of epithelial cells intoxicated by CN. CONCLUSIONS:The present observations should make us consider the potential interest of MB in the treatment of CN intoxication. The mechanisms of the antidotal properties of MB cannot be accounted for by the creation of a cyanomethemoglobinemia, rather its protective effects appears to be related to the unique properties of this redox dye, which, depending on the dose, could directly oppose some of the consequences of the metabolic depression produced by CN at the cellular level.
| S-EPMC6086742 | biostudies-literature
Project description:Breaking the lysosome helps its sequestered payloads access their molecular targets in cells and thus enhances the intracellular drug delivery. Current strategies for lysosomal escape involve direct physical interactions with the lipid membrane. These interactions pose a systemic toxicity and uncontrolled membrane rupture risk. Here, we report a light-detonated lysosome disruption using a hyaluronan (HA) nanogel packed with toludine blue (TB). The HA/TB nanogel is concentrated within the lysosomes. The applied light assists TB in generating reactive oxygen species and destroying the lysosome in situ, both in cells and isolated lysosomes. Real time fluorescent tracking reveals that quenched TB fluorescence recovers along with lysosome explosion, relocates to the nucleus, and is presented as a fluorescent sparkling in cells. This HA/TB, composed of all clinically approved materials, represents a biocompatible and facile strategy to "bomb" lysosomes in a spatiotemporally controlled fashion.
| S-EPMC6309271 | biostudies-literature
Project description:A promising alternative to antibiotics for treatment of Staphylococcus aureus infections is photodynamic inactivation (PDI), which employs a photosensitizer (PS) that produces cytotoxic reactive oxygen species (ROS) when exposed to molecular oxygen and antimicrobial blue light in the spectrum of 400-470 nm. Although the precise mechanistic basis of PDI has not been defined, the formation of ROS and free radicals that oxidize a number of cellular targets, including membrane lipids, damage to proteins and nucleic acids result in inactivation of essential cellular functions and subsequent cell death. Because PDI is non-selective and affects multiple cellular targets, development of resistance or tolerance to PDI has been considered to be unlikely and attempts to induce S. aureus resistance or tolerance upon repeated sub-lethal doses of PDI have not succeeded. However, multiple aspects of PDI suggest that development of tolerance is highly probable, in particular when PDI is used for treatment of infections where the environment at the infection site prevents penetration of PDI at a level sufficient to cause death of all bacteria and with tolerant phenotypes emerging from the surviving bacteria. In this study, we sought to identify the mechanisms that contribute to PDI tolerance in S. aureus. S. aureus HG003 and the isogenic HG003DmutSL strain with defects in DNA mismatch repair were used to evaluate the response of S. aureus and the roles of DNA mismatch repair and gene regulatory networks to repeated sublethal doses of PDI. Global transcriptome and genome analyses were used in an agnostic approach to identify the underlying transcriptional responses and genetic adaptations that occur as a result of repeated PDI and contribute to PDI tolerance. Our results reveal multiple metabolic, transport and cell wall biogenesis pathways that contribute to PDI tolerance, and a S. aureus regulatory gene likely responsible for the adaptive transcriptional response to PDI.
2020-07-01 | GSE153561 | GEO
Project description:ObjectiveWe previously demonstrated that upregulation of renal inducible nitric oxide synthase (iNOS) is associated with proximal tubule injury during systemic inflammation in humans. In this study we investigated the short-term effect of methylene blue (MB), an inhibitor of the NO pathway, on kidney damage and function in septic shock patients.Design and settingA prospective clinical study conducted in an intensive care unit.PatientsNine patients (four men, five women, mean age 71 +/- 3 years) with confirmed or suspected bacterial infection and with refractory septic shock defined as a mean arterial pressure < or = 70 mmHg despite norepinephrine infusion > or = 0.2 microg/kg per minute.InterventionsA 4 h continuous intravenous infusion of 1 mg/kg MB per hour.Measurements and resultsThe urinary excretion of NO metabolites decreased with median 90% (range 75-95%) from baseline to 6 h after MB administration. The first 24 h creatinine clearance improved by 51% (18-173%) after MB treatment but was still strongly impaired. During the first 6 h after the start of MB treatment both the urinary excretion of cytosolic glutathione S-transferase A1-1 and P1-1, markers for proximal and distal tubule damage, respectively, decreased by 45% (10-70%) and 70% (40-85) vs. baseline. After termination of the MB infusion the NO metabolites and markers of tubular injury returned to pretreatment levels.ConclusionsIn septic patients with refractory shock short-term infusion of MB is associated with a decrease in NO production and an attenuation of the urinary excretion of renal tubular injury markers.
| S-EPMC2228379 | biostudies-literature