ABSTRACT
Alzheimer's disease (AD) is a complex neurodegenerative disease that can lead to the loss of cognitive function. The progression of AD is regulated by multiple signaling pathways and their associated targets. Therefore, multitarget strategies theoretically have greater potential for treating AD. In this work, a series of new hybrids were designed and synthesized by the hybridization of tacrine (4, AChE: IC50 = 0.223 µM) with pyrimidone compound 5 (GSK-3ß: IC50 = 3 µM) using the cysteamine or cystamine group as the connector. The biological evaluation results demonstrated that most of the compounds exhibited moderate to good inhibitory activities against acetylcholinesterase (AChE) and glycogen synthase kinase 3ß (GSK-3ß). The optimal compound 18a possessed potent dual AChE/GSK-3ß inhibition (AChE: IC50 = 0.047 ± 0.002 µM, GSK-3ß: IC50 = 0.930 ± 0.080 µM). Further molecular docking and enzymatic kinetic studies revealed that this compound could occupy both the catalytic anionic site and the peripheral anionic site of AChE. The results also showed a lack of toxicity to SH-SY5Y neuroblastoma cells at concentrations of up to 25 µM. Collectively, this work explored the structure-activity relationships of novel tetrahydroacridin hybrids with sulfur-inserted linkers, providing a reference for the further research and development of new multitarget anti-AD drugs.
Subject(s)
Acetylcholinesterase , Alzheimer Disease , Cholinesterase Inhibitors , Drug Design , Glycogen Synthase Kinase 3 beta , Molecular Docking Simulation , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Humans , Cholinesterase Inhibitors/pharmacology , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Acetylcholinesterase/metabolism , Acetylcholinesterase/chemistry , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta/metabolism , Cell Line, Tumor , Sulfur/chemistry , Structure-Activity Relationship , Acridines/chemistry , Acridines/pharmacology , Acridines/chemical synthesis , Tacrine/chemistry , Tacrine/pharmacology , Tacrine/chemical synthesis , Molecular StructureABSTRACT
The classical neurotransmitter serotonin or 5-hydroxytryptamine (5-HT), synthesized from tryptophan, can be produced both centrally and peripherally. Through binding to functionally distinct receptors, serotonin is profoundly implicated in a number of fundamental physiological processes and pathogenic conditions. Recently, serotonin has been found covalently incorporated into proteins, a newly identified post-translational modification termed serotonylation. Transglutaminases (TGMs), especially TGM2, are responsible for catalyzing the transamidation reaction by transferring serotonin to the glutamine residues of target proteins. Small GTPases, extracellular matrix protein fibronectin, cytoskeletal proteins and histones are the most reported substrates for serotonylation, and their functions are triggered by this post-translational modification. This Review highlights the roles of serotonylation in physiology and diseases and provides perspectives for pharmacological interventions to ameliorate serotonylation for disease treatment.
Subject(s)
Monomeric GTP-Binding Proteins , Transglutaminases , Glutamine , Protein Processing, Post-Translational , Serotonin/metabolism , Transglutaminases/geneticsABSTRACT
Host-directed therapies are emerging as a promising tool in the curing of difficult-to-treat infections, such as those caused by drug-resistant bacteria. In this study, we aim to test the potential activity of the FDA- and EMA-approved drugs cysteamine and cystamine against Mycobacterium abscessus. In human macrophages (differentiated THP-1 cells), these drugs restricted M. abscessus growth similar to that achieved by amikacin. Here, we use the human ex vivo granuloma-like structures (GLS) model of infection with the M. abscessus rough (MAB-R) and smooth (MAB-S) variants to study the activity of new therapies against M. abscessus. We demonstrate that cysteamine and cystamine show a decrease in the number of total GLSs per well in the MAB-S and MAB-R infected human peripheral blood mononuclear cells (PBMCs). Furthermore, combined administration of cysteamine or cystamine with amikacin resulted in enhanced activity against the two M. abscessus morpho variants compared to treatment with amikacin only. Treatment with cysteamine and cystamine was more effective in reducing GLS size and bacterial load during MAB-S infection compared with MAB-R infection. Moreover, treatment with these two drugs drastically quenched the exuberant proinflammatory response triggered by the MAB-R variant. These findings showing the activity of cysteamine and cystamine against the R and S M. abscessus morphotypes support the use of these drugs as novel host-directed therapies against M. abscessus infections.
Subject(s)
Mycobacterium Infections, Nontuberculous , Mycobacterium abscessus , Humans , Amikacin/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Cysteamine/pharmacology , Cysteamine/therapeutic use , Cystamine/pharmacology , Cystamine/therapeutic use , Leukocytes, Mononuclear , Mycobacterium Infections, Nontuberculous/drug therapy , Mycobacterium Infections, Nontuberculous/microbiology , Microbial Sensitivity TestsABSTRACT
(1) Background: Radioprotective agents have garnered considerable interest due to their prospective applications in radiotherapy, public health medicine, and situations of large-scale accidental radiation exposure or impending radiological emergencies. Cystamine, an organic diamino-disulfide compound, is recognized for its radiation-protective and antioxidant properties. This study aims to utilize the aqueous ferrous sulfate (Fricke) dosimeter to measure the free-radical scavenging capabilities of cystamine during irradiation by fast carbon ions. This analysis spans an energy range from 6 to 500 MeV per nucleon, which correlates with "linear energy transfer" (LET) values ranging from approximately 248 keV/µm down to 9.3 keV/µm. (2) Methods: Monte Carlo track chemistry calculations were used to simulate the radiation-induced chemistry of aerated Fricke-cystamine solutions across a broad spectrum of cystamine concentrations, ranging from 10-6 to 1 M. (3) Results: In irradiated Fricke solutions containing cystamine, cystamine is observed to hinder the oxidation of Fe2+ ions, an effect triggered by oxidizing agents from the radiolysis of acidic water, resulting in reduced Fe3+ ion production. Our simulations, conducted both with and without accounting for the multiple ionization of water, confirm cystamine's ability to capture free radicals, highlighting its strong antioxidant properties. Aligning with prior research, our simulations also indicate that the protective and antioxidant efficiency of cystamine diminishes with increasing LET of the radiation. This result can be attributed to the changes in the geometry of the track structures when transitioning from lower to higher LETs. (4) Conclusions: If we can apply these fundamental research findings to biological systems at a physiological pH, the use of cystamine alongside carbon-ion hadrontherapy could present a promising approach to further improve the therapeutic ratio in cancer treatments.
Subject(s)
Cystamine , Linear Energy Transfer , Cystamine/pharmacology , Antioxidants , Radiation Dosimeters , Ions , Nucleons , Water/chemistry , CarbonABSTRACT
The application of fungicide mixture is one of the most important measures to extend the service life of highly selective fungicides. Pyraclostrobin (PYR), which has been extensively used to control plant diseases by inhibiting mitochondrial respiration of pathogenic fungi, is at a high risk of resistance development. In this study, the potential of PYR alone or in combination with cystamine, an inhibitor of microbial transglutaminase, to suppress Fusarium graminearum was tested in vitro and in vivo. A synergistic effect of PYR/CYS mixture was observed both in vitro and when applied to etiolated wheat coleoptile. The control effect of PYR/CYS mixture on F. graminearum was better than that of PYR alone, which was reflected by the increased protection effect. The discrepancies of membrane permeability and the redox-physiological state were observed between PYR and PYR/CYS treatments, suggesting that an increased PYR availability in F. graminearum mycelia could be related with the observed synergistic action. Moreover, a synergistic profile was observed between PYR and CYS in regard of massive autophagosomes in mycelia, indicating that enhanced autophagy could be involved in the mode of action of PYR/CYS mixture. The differential content of mitochondrial metabolites between PYR and PYR/CYS treatments also provided evidence for CYS contribution to the fungicidal action of PYR/CYS mixture. The results provide insight into the synergistic mechanism of action of PYR/CYS mixture and an effective way to enhance the efficiency of PYR to combat F. graminearum.
Subject(s)
Cystamine , Fungicides, Industrial , Autophagy , Fungicides, Industrial/pharmacology , PermeabilityABSTRACT
INTRODUCTION: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. METHODS: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. RESULTS: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. DISCUSSION/CONCLUSIONS: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.
Subject(s)
Arterial Pressure/drug effects , Cystamine/pharmacology , Enzyme Inhibitors/pharmacology , Hypertension, Pulmonary/prevention & control , Hypoxia/drug therapy , Protein Glutamine gamma Glutamyltransferase 2/antagonists & inhibitors , Pulmonary Artery/drug effects , Animals , Disease Models, Animal , Female , Hypertension, Pulmonary/enzymology , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/enzymology , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/physiopathology , Hypertrophy, Right Ventricular/prevention & control , Hypoxia/complications , Hypoxia/enzymology , Hypoxia/physiopathology , Male , Mice, Inbred C57BL , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Pulmonary Artery/enzymology , Pulmonary Artery/physiopathology , Pulmonary Fibrosis/enzymology , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/physiopathology , Pulmonary Fibrosis/prevention & control , Rats, Wistar , Vascular Remodeling/drug effects , Ventricular Function, Right/drug effects , Ventricular Remodeling/drug effectsABSTRACT
Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and nonenzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish, Pacifastacus leniusculus We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT, as well as from guinea pig, in a concentration-dependent manner. Cystamine injection decreased TGase activity in HPT without affecting production of reactive oxygen species. Moreover, the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.
Subject(s)
Astacoidea/enzymology , Astacoidea/physiology , Hematopoiesis , Transglutaminases/metabolism , Aggression , Animals , Astacoidea/drug effects , Behavior, Animal , Cystamine/pharmacology , Enzyme Inhibitors/pharmacology , Guinea Pigs , Hematopoiesis/drug effects , Male , Transglutaminases/antagonists & inhibitorsABSTRACT
A number of publications have reported that cysteamine has significant therapeutic effects on several aspects of Parkinson's disease (PD)-related pathology but none of these studies have evaluated its impact on pathological forms of α-Synuclein (α-Syn), one of the main hallmarks of PD. We therefore tested the efficacy of cysteamine on the Thy1-α-Syn mouse model which over-expresses full-length human wild-type α-Syn. Two-month (early stage disease) and 6-month old (late stage disease) mice and littermate controls were treated daily with cysteamine (20 mg/kg, i.p.) to assess the protective and restorative properties of this compound. After 6 weeks of treatment, animals were tested using a battery of motor tests. Cysteamine-treated transgenic mice displayed significant improvements in motor performance as compared to saline-treated transgenic littermates. Post-mortem readouts revealed a reduction in fibrillation, phosphorylation and total levels of overexpresed human α-Syn. To determine if such outcomes extended to human cells, the benefits of cysteamine were additionally tested using 6-hydroxydopamine (6-OHDA) treated neurons differentiated from induced pluripotent stem cells (iPSCs) derived from a PD patient harbouring a triplication of the SNCA gene. SNCA neurons treated with cysteamine exhibited significantly more intact/healthy neurites than cells treated with 6-OHDA alone. Additionally, SNCA neurons treated with cysteamine in the absence of 6-OHDA showed a trend towards lower total α-Syn levels. Overall, our in vivo and in vitro findings suggest that cysteamine can act as a disease-modifying molecule by enhancing -the survival of dopaminergic neurons and reducing pathological forms of α-Syn.
Subject(s)
Cysteamine/pharmacology , Dopaminergic Neurons/drug effects , Induced Pluripotent Stem Cells/drug effects , Parkinsonian Disorders/pathology , alpha-Synuclein/genetics , Animals , Dopaminergic Neurons/pathology , Humans , Locomotion/drug effects , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
The chemical cross-linking of complexes of proteins with nucleic acids is often used in structural and mechanistic studies of these oftentimes unstable and transient complexes. To date, no method has been reported for the thiol-based conjugation of proteins with an RNA backbone, mainly because of instability of the modified ribonucleic acid that is functionalized at the phosphodiester and its rapid hydrolysis. Here, we report the site-specific synthesis of stable RNA oligonucleotides with a thiol-bearing linker that was attached to the phosphodiester backbone, where the ribonucleotide at the cross-linking site was either replaced with 2'-deoxy- or 2'-fluororibonucleotide. The utility of this approach was validated in cross-linking tests with RNase H1, a model protein for RNA/DNA binding and key effector in DNA-like antisense drug therapy. Furthermore, scale-up cross-linking and purification of the complexes confirmed that the method is useful for obtaining preparations of protein-RNA/DNA complexes with purity and stability that are suitable for further biochemical and structural studies. The present approach broadens the repertoire of disulfide-based cross-linking strategies and is a novel tool for the stabilization of protein-RNA complexes in which the interaction occurs via the RNA backbone. This methodology may be broadly applicable to studies of otherwise unstable or transient complexes of proteins with RNA and RNA/DNA.
Subject(s)
RNA/metabolism , Ribonuclease H/metabolism , Base Sequence , Cross-Linking Reagents/chemistry , Cystamine/chemistry , Disulfides/chemistry , Humans , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Protein Binding , RNA/chemistry , Ribonuclease H/chemistry , Ribonuclease H/geneticsABSTRACT
This research demonstrated the development of a simple, cost-effective, and label-free immunosensor for the detection of α-synuclein (α-Syn) based on a cystamine (CYS) self-assembled monolayer (SAM) decorated fluorine-doped tin oxide (FTO) electrode. CYS-SAM was formed onto the FTO electrode by the adsorption of CYS molecules through the head sulfur groups. The free amine (-NH2) groups at the tail of the CYS-SAM enabled the immobilization of anti-α-Syn-antibody, which concurrently allowed the formation of immunocomplex by covalent bonding with α-Syn-antigen. The variation of the concentrations of the attached α-Syn at the immunosensor probe induced the alternation of the current and the charge transfer resistance (Rct) for the redox response of [Fe(CN)6]3-/4-, which displayed a linear dynamic range from 10 to 1000 ng/mL with a low detection limit (S/N = 3) of ca. 3.62 and 1.13 ng/mL in differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) measurements, respectively. The immunosensor displayed good reproducibility, anti-interference ability, and good recoveries of α-Syn detection in diluted human serum samples. The proposed immunosensor is a promising platform to detect α-Syn for the early diagnose of Parkinson's disease, which can be extended for the determination of other biologically important biomarkers.
Subject(s)
Biosensing Techniques/methods , Electrochemical Techniques/methods , Electrodes , Cystamine/chemistry , alpha-Synuclein/analysisABSTRACT
At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)-25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N'-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.
Subject(s)
Acrylamides/toxicity , Agmatine/toxicity , DNA/chemistry , Nuclear Localization Signals/pharmacology , Polyamines/toxicity , 3T3 Cells , Animals , Cell Line , Cell Membrane/physiology , Mice , Nuclear Localization Signals/chemistry , TransfectionABSTRACT
As a cationic non-viral gene delivery vector, poly(agmatine/ N, N'-cystamine-bis-acrylamide) (AGM-CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM-CBA/pDNA polyplexes was found to have a non-linear relationship with AGM-CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM-CBA), we used pGL3-control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM-CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate-limiting step for pLUC transfection expression. Further optimization of the non-viral gene delivery system can be focused on the improvement of gene intracellular availability.
Subject(s)
Cell Nucleus/metabolism , Genes, Reporter/genetics , Luciferases/genetics , Luciferases/metabolism , Plasmids/genetics , Transfection/methods , Acrylamides/chemistry , Agmatine/chemistry , Animals , Mice , NIH 3T3 Cells , Polyethyleneimine/chemistryABSTRACT
In this study, a surface chemical-modified rice husk biochar with abundant amino groups and disulfide bonds for the removal of cadmium was prepared using cystamine dihydrochloride as a modification ligand and glutaraldehyde as a crosslinker. The biochars were characterized by Fourier transform infrared spectrometry (FTIR), elemental analysis, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), thermogravimetry analysis (TGA), and nitrogen sorption (BET) before and after modification. The adsorption properties of the modified biochars for Cd (II) were investigated in detail via adsorption isotherm models, adsorption kinetics models, and selective adsorption experiments. The surfaces of the cystamine-modified biochars with granular nanopolymers of sufficient functional groups of primary amine and disulfide linkage rendered the biochar surface more conducive to electrostatic attraction and surface complexation. The theoretical maximum adsorption capacity of the modified biochars (81.02 mg g-1) was almost 10-fold greater than that of the raw biochars (8.347 mg g-1) for Cd (II). Besides, the cystamine-modified biochars had a better affinity for Cd (II) compared to other heavy metals (Zn, As, Cd, Co, Ni, Cr), showing six-fold greater affinity for Cd (II) than Zn2+. The results of this study indicate that the modification of biochars derived from rice husks shows great potential in the removal of Cd (II) from contaminated water.
Subject(s)
Cadmium/chemistry , Charcoal/chemistry , Water Pollutants, Chemical/chemistry , Water Purification , Kinetics , Oryza/chemistryABSTRACT
In this study, environmentally friendly, self-healing waterborne polyurethanes (WPUs) were prepared based on the disulfide metathesis reaction in cystamine. The cystamine acted as a chain extender in the WPU film, which showed a high mechanical strength of 19.1 MPa. The possibility of self-healing reaction was simultaneously modeled via liquid chromatography-mass spectrometry (LC-MS). WPU was confirmed to self-heal a surface crack thermally after a scratch test, and the efficiency was measured by comparing the mechanical properties before and after a cut-and-healing test. In addition, the disulfide-thiol exchange reaction was confirmed to occur in WPU with cystamine as a chain extender and 2-mercaptoethanol. Hot press tests confirmed the possibility of reprocessing the WPU. The WPU incorporating disulfide groups showed great potential as a smart self-healing material.
Subject(s)
Cystamine/chemistry , Polyurethanes/chemistry , Chromatography, Liquid , Disulfides/chemistry , Mass Spectrometry , Mechanical Phenomena , Polyurethanes/chemical synthesis , TemperatureABSTRACT
BACKGROUND: Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome-lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. OBJECTIVE: The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. RESULTS: We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTB-infected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. CONCLUSION: These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB.
Subject(s)
GTP-Binding Proteins/metabolism , Mycobacterium tuberculosis/pathogenicity , Transglutaminases/metabolism , Tuberculosis/metabolism , Animals , Autophagy , Blotting, Western , Cells, Cultured , Disease Models, Animal , Macrophages/metabolism , Macrophages/ultrastructure , Male , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Electron, Transmission , Protein Glutamine gamma Glutamyltransferase 2 , Tuberculosis/microbiology , Tuberculosis/pathologyABSTRACT
Tissue transglutaminase (TG2) plays an important role in pulmonary arterial hypertension (PAH). Previous research indicate that TG2 and protein serotonylation catalyzed by TG2 are upregulated in PAH. Serotonin transporter inhibitor fluoxetine ameliorates PAH via inhibition of protein serotonylation. It is still unknown whether PAH is inhibited through direct inhibition of TG2. Therefore, the present study aimed to investigate the effects of TG2 inhibitor cystamine on monocrotaline-induced PAH in rats. Rats were treated with monocrotaline (60 mg·kg-1, i.p.) in combination with or without cystamine (20, 40 mg·kg-1·day-1, p.o.). The results showed that compared with monocrotaline alone, combination of monocrotaline with cystamine (40 mg·kg-1·day-1, p.o.) relieved right ventricle hypertrophy, inhibited pulmonary arteriolar remodeling, and downregulated protein expression of TG2, phosphorylated protein kinase B (Akt), and extracellular regulated protein kinase (ERK) at day 21. However, except for TG2 expression, these changes were not significantly inhibited by cystamine at day 35. In addition, cystamine dose-dependently enhanced the survival rate of rats injected with monocrotaline at day 35. The findings suggest that cystamine slows but not reverses monocrotaline-induced PAH in rats, which was largely associated with the inhibition of TG2 protein expression and Akt and ERK activation.
Subject(s)
Cystamine/therapeutic use , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/drug therapy , Animals , Arterioles/pathology , Arterioles/physiopathology , Cystamine/pharmacology , Heart Septum/drug effects , Heart Septum/pathology , Heart Septum/physiopathology , Heart Ventricles/drug effects , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Hemodynamics/drug effects , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Lung/metabolism , Lung/pathology , Male , Monocrotaline , Pressure , Protein Glutamine gamma Glutamyltransferase 2 , Pulmonary Artery/drug effects , Pulmonary Artery/pathology , Pulmonary Artery/physiopathology , Rats, Sprague-Dawley , Serotonin Plasma Membrane Transport Proteins/metabolism , Signal Transduction/drug effects , Survival Analysis , Transglutaminases/metabolism , Vascular Remodeling/drug effectsABSTRACT
Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy.
Subject(s)
Cystamine/pharmacology , Golgi Apparatus/drug effects , Orexins/chemistry , Orexins/metabolism , Protein Aggregates/drug effects , Protein Aggregation, Pathological , Protein Disulfide-Isomerases/antagonists & inhibitors , Animals , Cells, Cultured , Cystamine/administration & dosage , Golgi Apparatus/metabolism , Hypothalamus , Male , Mice , Mice, Inbred C57BL , Neurons/drug effects , Neurons/metabolism , Protein Disulfide-Isomerases/metabolism , Rats , Rats, WistarABSTRACT
Activation of astrocytes has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Transglutaminase (TG) is a crosslinking enzyme and contributes to cell adhesion, cytoskeleton construct, extracellular matrix formation, and so on. One of the isozymes, tissue-type TG (TG2) is reported to be activated in AD. Moreover, amyloid ß1-42 (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be a substrate of TG2. However, contribution and derivation of TGs in brain for Aß aggregation remain to be clarified. In the present study, we examined the effects of cultured astrocytes prepared from rat embryonic brain cortex on Aß aggregation. When freshly prepared Aß was added to cultured astrocytes for 7 days, Aß monomer decreased and Aß oligomer unchanged. On the other hand, when Aß monomer was diluted with astrocytes conditioned medium, Aß oligomer increased time-dependently, and an inhibitor of TGs, cystamine, blocked it. Furthermore, when cultured astrocytes were stimulated with aggregated Aß, TG2 expression significantly increased. These results suggest that astrocytes could uptake Aß monomer to eliminate from brain; however, TGs derived from astrocytes might accelerate Aß aggregation and the aggregated Aß might enhance TG2 in astrocytes as a vicious cycle in pathological conditions. Adequate control of TGs expression and function in astrocytes would be an important factor in AD pathology.
Subject(s)
Amyloid beta-Peptides/metabolism , Astrocytes/metabolism , Peptide Fragments/metabolism , Protein Aggregation, Pathological/metabolism , Transglutaminases/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Astrocytes/drug effects , Cells, Cultured , Female , Peptide Fragments/pharmacology , Pregnancy , Protein Glutamine gamma Glutamyltransferase 2 , Rats , Rats, Wistar , Transglutaminases/isolation & purificationABSTRACT
Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.
Subject(s)
Amphotericin B/pharmacology , Endocytosis/physiology , GTP-Binding Proteins/biosynthesis , Gene Expression Regulation, Enzymologic , Microglia/enzymology , Transglutaminases/biosynthesis , Up-Regulation/physiology , Animals , Cell Line , Dose-Response Relationship, Drug , Endocytosis/drug effects , GTP-Binding Proteins/genetics , Mice , Microglia/drug effects , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/genetics , Up-Regulation/drug effectsABSTRACT
The therapeutic radiomitigating effect of cystamine and indralin was studied in experiments on mice and rats and pharmacological analysis of these drugs was carried out. The animals were subjected to whole-body 60Co γ-irradiation. The mice were exposed to single (9-10 Gy) or double (8 Gy) irradiation with an interval of 1 month. The rats were exposed to 10 Gy with partial shielding of the upper quarter of the abdomen. In experiments on mice, pretreatment with reserpine abolished the therapeutic effect of cystamine administered repeatedly every 15 min over 1 h after irradiation. Moreover, summation of the radioprotective and therapeutic effects of the radioprotector was revealed under these conditions. In mice and rats, α1-adrenoreceptor blocker terazosin did not abolish the therapeutic effect of indralin administrated after irradiation, but blocked the radioprotective effect of indralin applied prior to irradiation. At the same time, 5-HT2 serotonin receptor blocker tropoxin abolished the therapeutic effect of indralin without affecting its radioprotective activity.