Photo-excited Toluidine Blue Inhibits Full-Length Tau Aggregation in Alzheimer’ Disease

2019 ◽  
Author(s):  
Tushar Dubey ◽  
Nalini Gorantla ◽  
Kagepura Chandrashekara ◽  
Subashchandrabose Chinnathambi
Keyword(s):  
Toluidine Blue ◽  
Cell Biology ◽  
Cd Spectroscopy ◽  
Drosophila Model ◽  
Sds Page ◽  
Biophysical Analysis ◽  
Stressed Cells ◽  
Singlet Oxygen Species ◽  
Tau Aggregation

The aggregates of microtubule-associated protein Tau are the major hallmark of Alzheimer’sdisease. Tau aggregates accumulate intracellularly thus leading to generation of 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 irradiation with 630 nm light converted to photo-excited form leading to generation of singlet oxygen species. In present work we studied the potency of Toluidine blue and photo-excited Toluidine blue against Tau aggregation. Biochemical and biophysical analysis using ThSfluorescence, SDS-PAGE, CD spectroscopy and electron microscopy suggested that Toluidine blueinhibits the aggregation of Tau in-vitro. The Photo-excited toluidine blue potentially dissolved the matured Tau fibrils, which was indicating disaggregation property of Toluidine blue. The cell biology studies including cytotoxicity assays, ROS production assays suggested Toluidine blue to be a biocompatible dye as reduced ROS levels and cytotoxicity was observed after exposure of Toluidine blue on Tau stressed cells. The photo-excited Toluidine blue modulates the cytoskeleton network in cells, which was supported by immunofluorescence studies of neuronal cells. The studies in UAS Tau E14 transgenic Drosophila model suggested that photo-excited Toluidine blue was potent to restore the survival and memory deficit of Drosophila. The overall findings of our studies suggests that Toluidine blue to be a potent molecule in rescuing the Tau-mediated pathology by inhibiting its aggregation, reducing the cytotoxicity; modulating the tubulin level and behavioral characteristics of Drosophila. Thus Toluidine blue can be addressed as a potent molecule against Alzheimer’s disease.

Photo-excited Toluidine Blue Inhibits Full-Length Tau Aggregation in Alzheimer’ Disease

2019 ◽  
Author(s):  
Tushar Dubey ◽  
Nalini Gorantla ◽  
Kagepura Chandrashekara ◽  
Subashchandrabose Chinnathambi
Keyword(s):  
Toluidine Blue ◽  
Cell Biology ◽  
Cd Spectroscopy ◽  
Drosophila Model ◽  
Sds Page ◽  
Biophysical Analysis ◽  
Stressed Cells ◽  
Singlet Oxygen Species ◽  
Tau Aggregation

The aggregates of microtubule-associated protein Tau are the major hallmark of Alzheimer’sdisease. Tau aggregates accumulate intracellularly thus leading to generation of 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 irradiation with 630 nm light converted to photo-excited form leading to generation of singlet oxygen species. In present work we studied the potency of Toluidine blue and photo-excited Toluidine blue against Tau aggregation. Biochemical and biophysical analysis using ThSfluorescence, SDS-PAGE, CD spectroscopy and electron microscopy suggested that Toluidine blueinhibits the aggregation of Tau in-vitro. The Photo-excited toluidine blue potentially dissolved the matured Tau fibrils, which was indicating disaggregation property of Toluidine blue. The cell biology studies including cytotoxicity assays, ROS production assays suggested Toluidine blue to be a biocompatible dye as reduced ROS levels and cytotoxicity was observed after exposure of Toluidine blue on Tau stressed cells. The photo-excited Toluidine blue modulates the cytoskeleton network in cells, which was supported by immunofluorescence studies of neuronal cells. The studies in UAS Tau E14 transgenic Drosophila model suggested that photo-excited Toluidine blue was potent to restore the survival and memory deficit of Drosophila. The overall findings of our studies suggests that Toluidine blue to be a potent molecule in rescuing the Tau-mediated pathology by inhibiting its aggregation, reducing the cytotoxicity; modulating the tubulin level and behavioral characteristics of Drosophila. Thus Toluidine blue can be addressed as a potent molecule against Alzheimer’s disease.

scholarly journals Effect of Melatonin on Tau aggregation and Tau-mediated cell surface morphology

2019 ◽  
Author(s):  
Rashmi Das ◽  
Abhishek Ankur Balmik ◽  
Subashchandrabose Chinnathambi
Keyword(s):  
Cd Spectroscopy ◽  
Sem Analysis ◽  
Membrane Topology ◽  
Microtubule Assembly ◽  
Neuronal Protein ◽  
Transmission Electron ◽  
Tht Fluorescence ◽  
Melatonin Administration ◽  
Tau Aggregation

ABSTRACTTau is the major neuronal protein involved in the stabilization of microtubule assembly. In Alzheimer’s disease, Tau self assembles to form intracellular protein aggregates, which are toxic to cells. Various methods have been tried and tested to restrain the aggregation of Tau. Most of the agents tested for this purpose have limitations in their effectiveness and availability to neuronal cells. We tested melatonin against in vitro Tau aggregation and observed its effect on membrane topology, tubulin network and Tau phosphorylation in neuro2a and N9 cell lines. The aggregation and conformation of Tau was determined by ThT fluorescence and CD spectroscopy respectively. The morphology of Tau aggregates in presence and absence of melatonin was studied by transmission electron microscopy. Melatonin was found to reduce the formation of higher order oligomeric structures without affecting the overall aggregation kinetics of Tau. Melatonin also modulates and helps to maintain membrane topology as evidenced by FE-SEM analysis. Overall, melatonin administration shows mild anti-aggregation and cytoprotective effects.

Correlative microscopy in distrubuted (client/server) computing environments: tools for catalyzing the rapid dissemination of information about the cell biology of the human prostate

1995 ◽  
Vol 53 ◽  
pp. 682-683
Author(s):  
A. Hakam ◽  
J.T. Gau ◽  
M.L. Grove ◽  
B.A. Evans ◽  
M. Shuman ◽  
...  
Keyword(s):  
United States ◽  
Cell Biology ◽  
Tissue Bank ◽  
The United States ◽  
Prostate Adenocarcinoma ◽  
Correlative Microscopy ◽  
Client Server ◽  
Critical Elements ◽  
Transplant Organ

Prostate adenocarcinoma is the most common malignant tumor of men in the United States and is the third leading cause of death in men. Despite attempts at early detection, there will be 244,000 new cases and 44,000 deaths from the disease in the United States in 1995. Therapeutic progress against this disease is hindered by an incomplete understanding of prostate epithelial cell biology, the availability of human tissues for in vitro experimentation, slow dissemination of information between prostate cancer research teams and the increasing pressure to “ stretch” research dollars at the same time staff reductions are occurring.To meet these challenges, we have used the correlative microscopy (CM) and client/server (C/S) computing to increase productivity while decreasing costs. Critical elements of our program are as follows:1) Establishing the Western Pennsylvania Genitourinary (GU) Tissue Bank which includes >100 prostates from patients with prostate adenocarcinoma as well as >20 normal prostates from transplant organ donors.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

scholarly journals NeuroHeal Improves Muscle Regeneration after Injury

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 22
Author(s):  
Sara Marmolejo-Martínez-Artesero ◽  
David Romeo-Guitart ◽  
Vanesa Venegas ◽  
Mario Marotta ◽  
Caty Casas
Keyword(s):  
Satellite Cell ◽  
Muscle Regeneration ◽  
Cell Biology ◽  
Fiber Type ◽  
Traumatic Injury ◽  
Injury Rate ◽  
Scar Tissue ◽  
Medical Problem ◽  
Preclinical Model

Musculoskeletal injuries represent a challenging medical problem. Although the skeletal muscle is able to regenerate and recover after injury, the process engaged with conservative therapy can be inefficient, leading to a high re-injury rate. In addition, the formation of scar tissue implies an alteration of mechanical properties in muscle. There is still a need for new treatments of the injured muscle. NeuroHeal may be one option. Published studies demonstrated that it reduces muscle atrophy due to denervation and disuse. The main objective of the present work was to assess the potential of NeuroHeal to improve muscle regeneration after traumatic injury. Secondary objectives included characterizing the effect of NeuroHeal treatment on satellite cell biology. We used a rat model of sport-induced injury in the gastrocnemius and analyzed the effects of NeuroHeal on functional recovery by means of electrophysiology and tetanic force analysis. These studies were accompanied by immunohistochemistry of the injured muscle to analyze fibrosis, satellite cell state, and fiber type. In addition, we used an in vitro model to determine the effect of NeuroHeal on myoblast biology and partially decipher its mechanism of action. The results showed that NeuroHeal treatment advanced muscle fiber recovery after injury in a preclinical model of muscle injury, and significantly reduced the formation of scar tissue. In vitro, we observed that NeuroHeal accelerated the formation of myotubes. The results pave the way for novel therapeutic avenues for muscle/tendinous disorders.

scholarly journals Arrayed CRISPR reveals genetic regulators of tau aggregation, autophagy and mitochondria in Alzheimer’s disease model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lishu Duan ◽  
Mufeng Hu ◽  
Joseph A. Tamm ◽  
Yelena Y. Grinberg ◽  
Fang Shen ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Drug Discovery ◽  
Disease Model ◽  
Mitochondrial Morphology ◽  
Individual Gene ◽  
Human Genes ◽  
Future Drug ◽  
Tau Aggregation

AbstractAlzheimer’s disease (AD) is a common neurodegenerative disease with poor prognosis. New options for drug discovery targets are needed. We developed an imaging based arrayed CRISPR method to interrogate the human genome for modulation of in vitro correlates of AD features, and used this to assess 1525 human genes related to tau aggregation, autophagy and mitochondria. This work revealed (I) a network of tau aggregation modulators including the NF-κB pathway and inflammatory signaling, (II) a correlation between mitochondrial morphology, respiratory function and transcriptomics, (III) machine learning predicted novel roles of genes and pathways in autophagic processes and (IV) individual gene function inferences and interactions among biological processes via multi-feature clustering. These studies provide a platform to interrogate underexplored aspects of AD biology and offer several specific hypotheses for future drug discovery efforts.

scholarly journals Borrelidin from Saltern-Derived Halophilic Nocardiopsis sp. Dissociates Amyloid-β and Tau Fibrils

2020 ◽  
pp. 1-7
Author(s):  
Jisu Shin ◽  
Seung-Hoon Yang ◽  
Young Eun Du ◽  
Keunwan Park ◽  
DaWon Kim ◽  
...  
Keyword(s):  
Amyloid Β ◽  
Thioflavin T ◽  
Drug Candidate ◽  
Ht22 Cells ◽  
Aβ Aggregation ◽  
Actinomycete Strain ◽  
Direct Dissociation ◽  
Aβ Fibrils ◽  
Tau Aggregation

Background: Alzheimer’s disease (AD) is characterized by the aggregation of two pathological proteins, amyloid-β (Aβ) and tau, leading to neuronal and cognitive dysfunction. Clearance of either Aβ or tau aggregates by immunotherapy has become a potential therapy, as these aggregates are found in the brain ahead of the symptom onset. Given that Aβ and tau independently and cooperatively play critical roles in AD development, AD treatments might require therapeutic approaches to eliminate both aggregates together. Objective: We aimed to discover a chemical drug candidate from natural sources for direct dissociation of both insoluble Aβ and tau aggregates through in vitro assessments. Methods: We isolated four borrelidin chemicals from a saltern-derived halophilic actinomycete strain of rare genus Nocardiopsis and simulated their docking interactions with Aβ fibrils. Then, anti-cytotoxic, anti-Aβ, and anti-tau effects of borrelidins were examined by MTT assays with HT22 hippocampal cell line, thioflavin T assays, and gel electrophoresis. Results: When HT22 cells were exposed to Aβ aggregates, the treatment of borrelidins alleviates the Aβ-induced toxicity. These anti-cytotoxic effects can be derived from the inhibitory functions of borrelidins against the Aβ aggregation as shown in thioflavin T and gel electrophoretic analyses. Among them, especially borrelidin, which exhibits the highest probability of docking, not only dissociates Aβ aggregates but also directly regulates tau aggregation. Conclusion: Borrelidin dissociates insoluble Aβ and tau aggregates together and our findings support the view that it is possible to develop an alternative chemical approach mimicking anti-Aβ or anti-tau immunotherapy for clearance of both aggregates.

scholarly journals Cyclooxygenase Inhibition Alters Proliferative, Migratory, and Invasive Properties of Human Glioblastoma Cells In Vitro

2021 ◽  
Vol 22 (9) ◽  
pp. 4297
Author(s):  
Matthew Thomas Ferreira ◽  
Juliano Andreoli Miyake ◽  
Renata Nascimento Gomes ◽  
Fábio Feitoza ◽  
Pollyana Bulgarelli Stevannato ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Biology ◽  
Gelatin Zymography ◽  
Viable Cell ◽  
Cell Counting ◽  
Gelatinolytic Activity ◽  
Ep4 Receptor ◽  
And Migration ◽  
Proliferation And Migration

Prostaglandin E2 (PGE2) is known to increase glioblastoma (GBM) cell proliferation and migration while cyclooxygenase (COX) inhibition decreases proliferation and migration. The present study investigated the effects of COX inhibitors and PGE2 receptor antagonists on GBM cell biology. Cells were grown with inhibitors and dose response, viable cell counting, flow cytometry, cell migration, gene expression, Western blotting, and gelatin zymography studies were performed. The stimulatory effects of PGE2 and the inhibitory effects of ibuprofen (IBP) were confirmed in GBM cells. The EP2 and EP4 receptors were identified as important mediators of the actions of PGE2 in GBM cells. The concomitant inhibition of EP2 and EP4 caused a significant decrease in cell migration which was not reverted by exogenous PGE2. In T98G cells exogenous PGE2 increased latent MMP2 gelatinolytic activity. The inhibition of COX1 or COX2 caused significant alterations in MMP2 expression and gelatinolytic activity in GBM cells. These findings provide further evidence for the importance of PGE2 signalling through the EP2 and the EP4 receptor in the control of GBM cell biology. They also support the hypothesis that a relationship exists between COX1 and MMP2 in GBM cells which merits further investigation as a novel therapeutic target for drug development.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

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