RESUMO
Certain ligands slowly bind to acetylcholinesterase. As a result, there is a slow establishment of enzyme-inhibitor equilibrium characterized by a slow onset of inhibition prior reaching steady state. Three mechanisms account for slow-binding inhibition: a) slow binding rate constant kon, b) slow ligand induced-fit following a fast binding step, c) slow conformational selection of an enzyme form. The slow equilibrium may be followed by a chemical step. This later that can be irreversible has been observed with certain alkylating agents and substrate transition state analogs. Slow-binding inhibitors present long residence times on target. This results in prolonged pharmacological or toxicological action. Through several well-known molecules (e.g. huperzine) and new examples (tocopherol, trifluoroacetophenone and a 6-methyluracil alkylammonium derivative), we show that slow-binding inhibitors of acetylcholinesterase are promising drugs for treatment of neurological diseases such as Alzheimer disease and myasthenia gravis. Moreover, they may be of interest for neuroprotection (prophylaxis) against organophosphorus poisoning. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/enzimologia , Alcaloides/administração & dosagem , Alcaloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Animais , Humanos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Ligação Proteica , Sesquiterpenos/administração & dosagem , Sesquiterpenos/metabolismo , Tocoferóis/administração & dosagem , Tocoferóis/metabolismoRESUMO
Thirty to fifty percent of patients with acetylcholine receptor (AChR) antibody (Ab)-negative myasthenia gravis (MG) have Abs to muscle specific kinase (MuSK) and are referred to as having MuSK-MG. MuSK is a 100 kD single-pass post-synaptic transmembrane receptor tyrosine kinase crucial to the development and maintenance of the neuromuscular junction. The Abs in MuSK-MG are predominantly of the IgG4 immunoglobulin subclass. MuSK-MG differs from AChR-MG, in exhibiting more focal muscle involvement, including neck, shoulder, facial and bulbar-innervated muscles, as well as wasting of the involved muscles. MuSK-MG is highly associated with the HLA DR14-DQ5 haplotype and occurs predominantly in females with onset in the fourth decade of life. Some of the standard treatments of AChR-MG have been found to have limited effectiveness in MuSK-MG, including thymectomy and cholinesterase inhibitors. Therefore, current treatment involves immunosuppression, primarily by corticosteroids. In addition, patients respond especially well to B cell depletion agents, e.g., rituximab, with long-term remissions. Future treatments will likely derive from the ongoing analysis of the pathogenic mechanisms underlying this disease, including histologic and physiologic studies of the neuromuscular junction in patients as well as information derived from the development and study of animal models of the disease.
Assuntos
Músculos/patologia , Miastenia Gravis/enzimologia , Miastenia Gravis/patologia , Corticosteroides/uso terapêutico , Animais , Feminino , Subtipos Sorológicos de HLA-DR/genética , Haplótipos , Humanos , Imunoglobulina G/imunologia , Camundongos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Colinérgicos/genéticaRESUMO
Symptomatic treatment of myasthenia gravis is based on the use of peripherally-acting acetylcholinesterase (AChE) inhibitors that, in some cases, must be discontinued due to the occurrence of a number of side-effects. Thus, new AChE inhibitors are being developed and investigated for their potential use against this disease. Here, we have explored two alternative approaches to get access to peripherally-acting AChE inhibitors as new agents against myasthenia gravis, by structural modification of the brain permeable anti-Alzheimer AChE inhibitors tacrine, 6-chlorotacrine, and huprine Y. Both quaternization upon methylation of the quinoline nitrogen atom, and tethering of a triazole ring, with, in some cases, the additional incorporation of a polyphenol-like moiety, result in more polar compounds with higher inhibitory activity against human AChE (up to 190-fold) and butyrylcholinesterase (up to 40-fold) than pyridostigmine, the standard drug for symptomatic treatment of myasthenia gravis. The novel compounds are furthermore devoid of brain permeability, thereby emerging as interesting leads against myasthenia gravis.
Assuntos
Acetilcolinesterase/metabolismo , Aminoacridinas/síntese química , Aminoquinolinas/síntese química , Inibidores da Colinesterase/síntese química , Acetilcolinesterase/química , Aminoacridinas/química , Aminoacridinas/farmacologia , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Regulação para Baixo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Modelos Moleculares , Estrutura Molecular , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Relação Estrutura-Atividade , Tacrina/químicaRESUMO
Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.
Assuntos
Autofagia , Anidrase Carbônica III/metabolismo , Endocitose , Miastenia Gravis/enzimologia , Receptores Nicotínicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Muscle-specific kinase (MuSK) myasthenia gravis (MG) is hallmarked by the predominant involvement of bulbar muscles and muscle atrophy. This might mimic amyotrophic lateral sclerosis (ALS) presenting with bulbar weakness. We encountered four cases of MuSK MG patients with an initial misdiagnosis of ALS. We analyzed the clinical data of the four misdiagnosed MuSK MG patients, and investigated the presence of MuSK autoantibodies in a group of 256 Dutch bulbar-onset ALS patients using a recombinant MuSK ELISA and a standard MuSK radioimmunorecipitation assay. Clues for changing the diagnosis were slow progression, clinical improvement, development of diplopia and absence of signs of upper motor neuron involvement. No cases of MuSK MG were identified among a group of 256 bulbar ALS patients diagnosed according to the revised El Escorial criteria. A misdiagnosis of ALS in patients with MuSK MG is rare. We recommend to carefully consider the diagnosis of MuSK MG in patients presenting with bulbar weakness without clear signs of upper motor neuron dysfunction.
Assuntos
Autoanticorpos/sangue , Miastenia Gravis/diagnóstico , Miastenia Gravis/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Idoso , Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores/sangue , Erros de Diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologiaRESUMO
The low-density lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific receptor tyrosine kinase (MuSK) form a tetrameric protein complex on the postsynaptic membrane at the neuromuscular junction (NMJ). Binding of agrin to LRP4 triggers phosphorylation of MuSK. Activated MuSK drives clustering of acetylcholine receptor (AChR). Wnt ligands also directly bind to MuSK to induce AChR clustering. MuSK anchors the acetylcholinesterase (AChE)/collagen Q (ColQ) complex to the synaptic basal lamina. In addition, an extracellular proteoglycan, biglycan, binds to MuSK. Anti-MuSK autoantibodies (MuSK-IgG) are observed in 5-15% of autoimmune myasthenia gravis (MG) patients. MuSK-IgG blocks both ColQ-MuSK and LRP4-MuSK interactions. MuSK-IgG, LRP4, ColQ, and biglycan bind to the immunoglobulin-like domains 1 and 4 of MuSK. Lack of the effects of cholinesterase inhibitors in MuSK-MG patients is likely due to hindrance of ColQ-MuSK interaction by MuSK-IgG and subsequent deficiency of AChE observed in model mice, which, however, has not been proven in MuSK-MG patients. As ColQ enhances expression of membrane-bound MuSK, inhibition of ColQ-MuSK interaction by MuSK-IgG may account for lack of AChR clusters in MuSK-MG. We thus made passive transfer models using Colq+/+ and Colq-/- mice to dissect the effect of ColQ on AChR clustering in MuSK-MG. We found that MuSK-IgG-mediated suppression of LRP4-MuSK interaction, not of ColQ-MuSK interaction, caused defective AChR clustering. We also unexpectedly observed that both MuSK-IgG and ColQ suppressed agrin/LRP4/MuSK signaling in dose-dependent manners. Quantitative comparison revealed that MuSK-IgG blocked agrin-LRP4-MuSK signaling more than ColQ. We propose that attenuation of AChR clustering in MuSK-MG is due to hindrance of LRP4-MuSK interaction in the presence of agrin by MuSK-IgG.
Assuntos
Anticorpos/metabolismo , Colágeno/metabolismo , Miastenia Gravis/enzimologia , Miastenia Gravis/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Animais , Humanos , Junção Neuromuscular/metabolismo , Ligação Proteica , Receptores Proteína Tirosina Quinases/químicaRESUMO
Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme·inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 µM; Ki'=3.17 µM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 Å resolution. The complex is stabilized by cation-π, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, τ=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.
Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/química , Simulação de Acoplamento Molecular , Miastenia Gravis , Compostos de Amônio Quaternário/química , Uracila/análogos & derivados , Animais , Células CHO , Inibidores da Colinesterase/uso terapêutico , Cricetinae , Cricetulus , Humanos , Camundongos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Compostos de Amônio Quaternário/uso terapêutico , Uracila/química , Uracila/uso terapêuticoRESUMO
Slow-binding inhibition (SBI) of enzymes is characterized by slow establishment of enzyme-inhibitor equilibrium. Cholinesterases (ChEs) display slow onset of inhibition with certain inhibitors. After a survey of SBI mechanisms, SBI of ChEs is examined. SBI results either from simple slow interaction, induced-fit, or slow conformational selection. In some cases, the slow equilibrium is followed by an irreversible chemical step. This later was observed for the interaction of ChEs with certain irreversible inhibitors. Because slow-binding inhibitors present pharmacological advantages over classical reversible inhibitors (e.g. long target-residence times, resulting in prolonged efficacy with minimal unwanted side effects), slow-binding inhibitors of ChEs are promising new drugs for treatment of Alzheimer disease, myasthenia, and neuroprotection. SBI is also of toxicological importance; it may play a role in mechanisms of resistance and protection against poisoning by irreversible agents.
Assuntos
Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/toxicidade , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Animais , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/uso terapêutico , Humanos , Modelos Moleculares , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Fármacos Neuroprotetores/toxicidade , Ligação Proteica , Conformação ProteicaRESUMO
Myasthenia gravis (MG) is a neuromuscular disease that is conventionally treated with acetylcholinesterase (AChE) inhibitors, which may not fully remove the symptom for many reasons. When AChE inhibitors do not work, Chinese patients turn to Chinese medicine, such as the Buzhongyiqi decoction (BD), to treat MG. By elucidating the relations between the herbs of the Buzhongyiqi decoction recipe and AChE inhibitors with structure-based and ligand-based drug design methods and chemoinformatics approaches, we have found the key active components of BD. Using these key active components as templates, we have discovered five new AChE inhibitors through virtual screening of a commercial compound library. The new AChE inhibitors have been confirmed with Ellman assays. This study demonstrates that lead identification can be inspired by elucidating Chinese medicine. Since BD is a mixture, further studies against other drug targets are needed.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Descoberta de Drogas , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologiaRESUMO
AIM: Muscle specific kinase (MuSK) antibody-positive myasthenia gravis(MG) patients might present with clinical and electrophysiological signs of muscle atrophy. In this study, we investigated the potential contribution of mitochondrial dysfunction to muscle atrophy induced by MuSK immunity. METHODS: Mitochondrial enzyme expression was investigated in muscle samples of MuSK-immunized, acetylcholine receptor (AChR)-immunized, and complete Freund's adjuvant (CFA)-immunized C57BL/6 (B6) mice using histochemical methods. Mitochondrial enzyme activity was also investigated in MuSK- and CFA-immunized mice. RESULTS: Histochemical analysis showed normal muscle fiber activity on succinate dehydrogenase (SDH) and cytochrome oxidase (COX) stains in all immunization groups. However, MuSK-immunized mice had more ragged-red fibers on modified Gomori-trichrome (MGT) stain and more pronounced type 1 muscle fiber atrophy. MuSK-immunized mice also showed reduced citrate synthase, SDH, and NADH-cytochrome c-reductase activity. DISCUSSION: Our results suggest that MuSK-immunity might induce muscle atrophy through mitochondrial dysfunction.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Doenças Musculares/enzimologia , Miastenia Gravis/enzimologia , Fosfotransferases/imunologia , Succinato Desidrogenase/metabolismo , Animais , Autoanticorpos/imunologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes.
Assuntos
AMP Desaminase/metabolismo , Miastenia Gravis/enzimologia , Timo/enzimologia , AMP Desaminase/genética , Adulto , Idoso , Ativação Enzimática , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/genética , Miastenia Gravis/patologia , Timo/patologiaRESUMO
AMP dephosphorylation via ecto-5'-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5'-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4(+)CD25(+)FoxP3(+) regulatory T cells express lower amounts of ecto-5'-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4(+) T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5'-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Desaminase/sangue , Adenosina/metabolismo , Miastenia Gravis/enzimologia , Miastenia Gravis/metabolismo , 5'-Nucleotidase/deficiência , Adenosina Desaminase/deficiência , Animais , Feminino , Contração Muscular/genética , Contração Muscular/imunologia , Contração Muscular/fisiologia , Miastenia Gravis/imunologia , Ratos , Ratos Wistar , Transmissão Sináptica/genética , Linfócitos T Reguladores/metabolismoRESUMO
Muscle-specific kinase (MuSK) belongs to the nicotinic acetylcholine receptor complex which is targeted by pathogenic autoantibodies causing Myasthenia gravis. While up to 95% of patients with generalized Myasthenia gravis were shown to be positive for acetylcholine receptor-specific autoantibodies, up to 70% of the remaining patients develop autoantibodies against MuSK. Discrimination of the autoantibody specificity is important for therapy of Myasthenia gravis. Recently, the new automatic fluorescence assessment platform AKLIDES has been developed for immunofluorescence-based diagnostics of autoimmune diseases. In order to establish an AKLIDES procedure for the detection of MuSK-specific autoantibodies (anti-MuSK), we developed a recombinant HEp-2 cell clone expressing the human MuSK cDNA. Here we show at the mRNA and protein level that the cell clone HEp-2 M4 stably expresses human MuSK. We provide evidence for a localization of MuSK at the cell membrane. Using cell clone HEp-2 M4 on the AKLIDES system, we investigated 34 patient sera that were previously tested anti-MuSK positive by radioimmunoassay as positive controls. As negative controls, we tested 29 acetylcholine receptor-positive but MuSK-negative patient sera, 30 amytrophic lateral sclerosis (ALS) patient sera and 45 blood donors. HEp-2 M4 cells revealed a high specificity for the detection of MuSK autoantibodies from 25 patient sera assessed by a specific pattern on HEp-2 M4 cells. By using appropriate cell culture additives, the fraction of cells stained positive with anti-MuSK containing sera can be increased from 2-16% to 10-48%, depending on the serum. In conclusion, we provide data showing that the novel recombinant cell line HEp-2 M4 can be used to screen for anti-MuSK with the automatic AKLIDES system.
Assuntos
Miastenia Gravis/diagnóstico , Miastenia Gravis/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo , Autoanticorpos/sangue , Autoanticorpos/imunologia , Automação , Linhagem Celular , Proliferação de Células , Forma Celular , Imunofluorescência , Humanos , Miastenia Gravis/sangue , Miastenia Gravis/imunologia , Especificidade de ÓrgãosRESUMO
BACKGROUND: To investigate the expression and clinical significance of protein tyrosine phosphatase nonreceptor 22 (PTPN22) in thymoma, as well as the relationship between thymoma and myasthenia gravis (MG). METHODS: The expression of PTPN22 in normal thymus (35 cases), thymoma without MG (50 cases), and thymoma with MG (45 cases) treated with surgery were detected by the EnVision two-step immunohistochemical staining method. We analyzed the relationship of thymoma with MG as well as some clinical factors, such as the Osserman classification for MC, age, gender, and course of disease before surgery. All data were analyzed using the SPSS software. RESULTS: The positive rates of PTPN22 expression in thymoma with and without MG were 11.1% (5/45) and 24.0% (12/50), respectively, which were significantly lower than in the normal thymus (74.3%, 26/35). A significant difference was found between the positive rates of thymoma with and without MG. The expression level of PTPN22 had no relation to thymoma with MG and to the Osserman classification for MG, age, gender, and course of disease of the patient. CONCLUSIONS: Minimal or no PTPN22 expression in thymoma may play an important role in the development of thymoma with MG, provide basis for further genetic research.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Timoma/enzimologia , Neoplasias do Timo/enzimologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Miastenia Gravis/complicações , Miastenia Gravis/enzimologia , Timoma/complicações , Timoma/cirurgia , Neoplasias do Timo/complicações , Neoplasias do Timo/cirurgiaRESUMO
OBJECTIVE: To investigate the protective role of Sijunzi decoction in neuromuscular junction (NMJ) and muscle cell mitochondria ultrastructure; as well as its effects on the amount of adenosine triphosphate (ATP) and the activities of mitochondrial respiratory chain complexes I, II, III, and IV in autoimmune myasthenia gravis rats. METHODS: An experimental autoimmune myasthenia gravis (EAMG) rat model was established by inoculating rats with acetylcholine receptors extracted from Torpedo. Rats were divided into three groups: model, prednisone, and Sijunzi decoction, and were fed physiological saline, prednisone, or Sijunzi decoction, respectively. NMJ and muscle cell mitochondria ultrastructure were observed by transmission electron microscope. The amount of ATP was assessed by high performance liquid chromatography. The activities of mitochondrial respiratory chain complexes I, II, III, and IV was determined using the Clark oxygen electrode method. RESULTS: In the model group, there were sparse muscle fibers, with decreased mitochondria, and sparse, diffluent, or absent NMJ folds. After intervention with Sijunzi decoction, the above pathology changes were improved: muscle fiber structure was clear and complete; the mitochondria count was higher; and the NMJ structure was close to normal. Gastrocnemius muscle mitochondria in the model group produced significantly less ATP than those in the prednisone group (P < 0.01). Conversely, the ATP of Sijunzi decoction group was significantly higher than prednisone group (P < 0.01). The activities of gastrocnemius muscle mitochondrial respiratory chain complexes I, II, III, and IV in both the prednisone and Sijunzi decoction groups was dramatically higher compared with the model group (P < 0.05). The activities of complexes I and III in the Sijunzi decoction group were significantly higher than those in the prednisone group (P < 0.05), but there was no obvious difference in complex II or IV activities between the two groups (P > 0.05). CONCLUSION: Sijunzi decoction improved pathological changes in muscle mitochondria and NMJ, enhanced the amount of ATP in gastrocnemius muscle mitochondria, and improved the activities of respiratory chain complexes I, II, III, and IV (especially I and III) of the EAMG rats.
Assuntos
Medicamentos de Ervas Chinesas/administração & dosagem , Miastenia Gravis/tratamento farmacológico , Junção Neuromuscular/efeitos dos fármacos , Substâncias Protetoras/administração & dosagem , Trifosfato de Adenosina/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Feminino , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Miastenia Gravis/enzimologia , Miastenia Gravis/metabolismo , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Ratos , Ratos Endogâmicos LewRESUMO
We herein report a case of ocular myasthenia gravis (MG) that was highly positive for anti-muscle-specific tyrosine kinase (MuSK) antibodies. The examined patient exhibited bilateral ptosis and lateral gaze palsy without any generalized symptoms and was diagnosed with ocular MG with anti-MuSK antibodies. She responded to treatment with prednisolone and immunosuppressants and experienced only ocular symptoms for four years and eight months after onset. Ocular MG with anti-MuSK antibodies lasting for a long term has rarely been described. Our findings suggest that it may be reasonable to test for the presence of anti-MuSK antibodies in patients who present with external ophthalmoplegia.
Assuntos
Autoanticorpos/sangue , Oftalmopatias/imunologia , Miastenia Gravis/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Adulto , Oftalmopatias/complicações , Oftalmopatias/diagnóstico , Oftalmopatias/enzimologia , Feminino , Humanos , Miastenia Gravis/complicações , Miastenia Gravis/diagnóstico , Miastenia Gravis/enzimologia , Oftalmoplegia/etiologia , Oftalmoplegia/imunologia , Fatores de TempoRESUMO
INTRODUCTION: Both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are present in the body in large amounts. AChE is an important part of the cholinergic nervous system taking place in the central and peripheral nervous system. AChE is a target of several toxins such as insecticide carbofuran, nerve agents, sarin, soman, tabun and VX. Beside toxins, drugs for treatment of Alzheimer's disease and myasthenia gravis, such as galantamine, donepezil, rivastigmine, tacrine, huperzine, pyridostigmine and neostigmine, are known. AREAS COVERED: The review gives an overview of the importance of the cholinergic nervous system, the biochemistry of AChE and the role of AChE inhibitors. Current efforts to introduce potent drugs for Alzheimer's disease therapy and reduce toxicity, while keeping the maximal pharmacological effect, are also discussed. EXPERT OPINION: The current research effort into AChE inhibitors can be divided into two categories. First, new toxins useful for agricultural purposes and second, novel drugs that need to be prepared, although there is less interest in the new toxins. The research for drugs for Alzheimer's disease needs to focus on inhibitors that reduce the deposition of amyloid plaques, but do not initiate AChE expression.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Acetilcolina/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Animais , Química Farmacêutica , Inibidores da Colinesterase/efeitos adversos , Inibidores da Colinesterase/química , Desenho de Fármacos , Humanos , Legislação de Medicamentos , Estrutura Molecular , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Patentes como Assunto , Relação Estrutura-AtividadeRESUMO
It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one 'activator' domain activates the other 'receiver' domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.
Assuntos
Citoplasma/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Citoplasma/metabolismo , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Terapia de Alvo Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Miastenia Gravis/genética , Mioblastos/enzimologia , Mioblastos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Junção Neuromuscular/enzimologia , Junção Neuromuscular/metabolismo , Ligação ProteicaRESUMO
Autoimmune myasthenia gravis is usually characterized by the presence of autoantibodies against the acetylcholine receptor (~80-90% of patients) or muscle-specific tyrosine kinase (MuSK) (~5% of patients). In the remaining patients, no such antibodies (Abs) are detectable, but this could be due either to the presence of auto-Abs to yet unidentified antigens or to concentrations of circulating Abs below the threshold of the available assays. The most popular and sensitive assay for anti-MuSK Abs is a radioimmunoprecipitation assay (RIPA), which uses (125)I-labeled MuSK as test antigen. A serious limiting factor of the sensitivity of such RIPAs is that small volumes of test serum are required (maximum 5-20 µl) in order to avoid excessive background values. We have overcome this obstacle by the development of a two-step RIPA. This involves non-stringent affinity purification of anti-MuSK Abs from a large volume of patient's serum, followed by a regular RIPA step, with the isolated Abs, to determine the antibody titer. This two-step assay was shown to be 10-50 times more sensitive than the regular RIPA. All tested sera previously found to be positive in the regular RIPA and normal sera were also positive or negative in the two-step RIPA, respectively. Importantly, of seven tested sera previously characterized by regular RIPA as negative with titers that were above zero, but statistically not significantly higher from the background, two were found to be positive, while the others were clearly shown to be negative. We conclude that our diagnostic test can detect very low concentrations of circulating anti-MuSK Abs. The general principle of this two-step RIPA approach may also be applied to the detection of currently undetectable concentrations of circulating auto-Abs involved in other diseases.
Assuntos
Autoanticorpos/biossíntese , Miastenia Gravis/imunologia , Miastenia Gravis/terapia , Ensaio de Radioimunoprecipitação/métodos , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/sangue , Autoantígenos/imunologia , Glicosilação , Humanos , Miastenia Gravis/enzimologia , Ensaio de Radioimunoprecipitação/instrumentação , Ensaio de Radioimunoprecipitação/normas , Receptores Proteína Tirosina Quinases/sangue , Receptores Colinérgicos/sangue , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Myasthenia gravis (MG) is an autoimmune disease with weakness in striated musculature due to anti-acetylcholine receptor (AChR) antibodies or muscle specific kinase at the neuromuscular junction. A subgroup of patients has periocular symptoms only; ocular MG (OMG). Matrix metalloproteinases (MMP) are increased in several autoimmune diseases, including generalized MG (GMG), and have been suggested to play a role in immune cell infiltration, basement membrane breakdown and autoimmune pathogenesis. METHODS: Total levels of MMP2, MMP3 and MMP9 were measured in serum by ELISA. RESULTS: The MG patients had increased serum levels of MMP2 (median values 200.7 vs. 159.7 ng/ml, p < 0.001) and MMP9 (median values 629.6 vs. 386.4 ng/ml, p < 0.001) compared to controls. A subgroup of patients had increased MMP3 concentration (p = 0.001). The differences were not dependent on presence of AChR antibodies. No difference was observed between GMG and OMG patients with regard to MMP2 (p = 0.598), MMP3 (p = 0.450) and MMP9 (p = 0.271). DISCUSSION: The increased MMP levels in our MG patients group and the lack of dependence on anti-AChR antibodies suggest that MMP2, MMP3 and MMP9 play a role in the development of MG. The similarities between GMG and OMG support OMG as a systemic disease.