RESUMEN
Actin-interacting proteins are important molecules for filament assembly and cytoskeletal signaling within vascular endothelium. Disruption in their interactions causes endothelial pathogenesis through redox imbalance. Actin filament redox regulation remains largely unexplored, in the context of pharmacological treatment. This work focused on the peptidyl methionine (M) redox regulation of actin-interacting proteins, aiming at elucidating its role on governing antioxidative signaling and response. Endothelial EA.hy926 cells were subjected to treatment with salvianolic acid B (Sal B) and tert-butyl-hydroperoxide (tBHP) stimulation. Mass spectrometry was employed to characterize redox status of proteins, including actin, myosin-9, kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (Keap1), plastin-3, prelamin-A/C and vimentin. The protein redox landscape revealed distinct stoichiometric ratios or reaction site transitions mediated by M sulfoxide reductase and reactive oxygen species. In comparison with effects of tBHP stimulation, Sal B treatment prevented oxidation at actin M325, myosin-9 M1489/1565, Keap1 M120, plastin-3 M592, prelamin-A/C M187/371/540 and vimentin M344. For Keap1, reaction site was transitioned within its scaffolding region to the actin ring. These protein M oxidation regulations contributed to the Sal B cytoprotective effects on actin filament. Additionally, regarding the Keap1 homo-dimerization region, Sal B preventive roles against M120 oxidation acted as a primary signal driver to activate nuclear factor erythroid 2-related factor 2 (Nrf2). Transcriptional splicing of non-POU domain-containing octamer-binding protein was validated during the Sal B-mediated overexpression of NAD(P)H dehydrogenase [quinone] 1. This molecular redox regulation of actin-interacting proteins provided valuable insights into the phenolic structures of Sal B analogs, showing potential antioxidative effects on vascular endothelium.
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Actinas , Antioxidantes , Benzofuranos , Depsidos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Actinas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Vimentina/metabolismo , Estrés Oxidativo , Metionina , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Proteínas del Citoesqueleto/metabolismo , Miosinas/metabolismo , Miosinas/farmacologíaRESUMEN
OBJECTIVES: To determine the role of MYL4 regulation of lysosomal function and its disturbance in fibrotic atrial cardiomyopathy. BACKGROUND: We have previously demonstrated that the atrial-specific essential light chain protein MYL4 is required for atrial contractile, electrical, and structural integrity. MYL4 mutation/dysfunction leads to atrial fibrosis, standstill, and dysrhythmia. However, the underlying pathogenic mechanisms remain unclear. METHODS AND RESULTS: Rats subjected to knock-in of a pathogenic MYL4 mutant (p.E11K) developed fibrotic atrial cardiomyopathy. Proteome analysis and single-cell RNA sequencing indicate enrichment of autophagy pathways in mutant-MYL4 atrial dysfunction. Immunofluorescence and electron microscopy revealed undegraded autophagic vesicles accumulated in MYL4p.E11K rat atrium. Next, we identified that dysfunctional MYL4 protein impairs autophagy flux in vitro and in vivo. Cardiac lysosome positioning and mobility were regulated by MYL4 in cardiomyocytes, which affected lysosomal acidification and maturation of lysosomal cathepsins. We then examined the effects of MYL4 overexpression via adenoviral gene-transfer on atrial cardiomyopathy induced by MYL4 mutation: MYL4 protein overexpression attenuated atrial structural remodeling and autophagy dysfunction. CONCLUSIONS: MYL4 regulates autophagic flux in atrial cardiomyocytes via lysosomal mobility. MYL4 overexpression attenuates MYL4 p.E11K induced fibrotic atrial cardiomyopathy, while correcting autophagy and lysosomal function. These results provide a molecular basis for MYL4-mutant induced fibrotic atrial cardiomyopathy and identify a potential biological-therapy approach for the treatment of atrial fibrosis.
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Fibrilación Atrial , Cardiomiopatías , Cadenas Ligeras de Miosina , Animales , Ratas , Fibrilación Atrial/metabolismo , Autofagia/genética , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Fibrosis , Lisosomas/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Miosinas/metabolismo , Miosinas/farmacologíaRESUMEN
The effects of mechanical stress on cultured muscle cells were examined with particular interest in myofibril assembly by using a cell-stretching system. We observed that formation and maintenance of cross-striated myofibrils in chick muscle cell cultures was suppressed in the media containing higher concentration of KCl, tetrodotoxin, or ML-9 (an inhibitor of myosin light chain kinase), but periodic stretching of myotubes for several days enabled formation of striated myofibrils just as in standard muscle cultures. However, ryanodine (a blocker of the Ca2 + channel in sarcoplasmic reticulum) and BDM (an inhibitor of myosin-actin interaction) suppressed the stretch-induced myofibrillogenesis. We further found that stretching of myotubes causes quick and transient elevation of the intracellular Ca2 + concentration and this elevation is disturbed by inhibition of Ca2 + channels of sarcoplasmic reticulum and suppression of Ca2 + influx from culture medium. These observations indicate that periodic stretching induces elevation of intracellular Ca2 + concentration and that this elevation may be due to release of Ca2 + from sarcoplasmic reticulum and Ca2 + influx from outside of the cells. The increased Ca2 + may activate actin-myosin interaction by interacting with troponin that is located along actin filaments and/or inducing phosphorylation of myosin light chains and thereby promote myofibril assembly.
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Actinas , Miofibrillas , Animales , Células Cultivadas , Desarrollo de Músculos , Fibras Musculares Esqueléticas , Miosinas/farmacologíaRESUMEN
BACKGROUND: Ancient prescriptions of Suo Quan Wan (SQW) have therapeutic effects on diabetic bladder dysfunction. However, the underlying mechanism remains unclear. Here, we hypothesized that SQW ameliorates bladder overactivity and regulates neurotransmission via regulating Myosin Va protein expression. METHODS: After diabetic rats were induced by streptozotocin (65 mg/kg), the model of diabetic bladder dysfunction was established by detecting fasting blood glucose, urodynamic test, in vitro muscle strip experiments, and histological examination. One week after induction, SQW was given to observe the therapeutic effect. The expression levels of Myosin Va in control, Model, SQW L and SQW H groups were detected by RT-qPCR, RNAscope and immunofluorescence assay. The expression levels of ChAT, SP, nNOS and VIP proteins were observed by immunofluorescence assay. After knockdown and overexpression of Myosin Va, the expression changes of ChAT, SP, nNOS and VIP and the regulatory role of SQW were observed. RESULTS: STZ-induced DM rats had significantly higher serum glucose levels and lower body weight. Compared with the diabetic rats, SQW treatment significantly improved urination function with decreased residual volume (RV), bladder compliance (BC), non-voiding contractions (NVCs), and increased voided efficiency (VE). In addition, contractile responses of muscle strips to electrical-field stimulation (EFS), carbachol (CCh), KCl were significantly lower in the SQW H and SQW L groups than those in the model group. RT-qPCR found that the expression of Myosin Va in the bladder tissue or bladder neurons in model group was significantly increased compared with the control group, and SQW treatment significantly decreased the levels of Myosin Va. In DM rats, ChAT and SP expression were significantly increased, while nNOS and VIP expression were significantly decreased, and SQW improved this phenomenon. Interestingly, SQW ameliorated the abnormal expression of ChAT, SP, nNOS and VIP caused by myosin Va knockdown, and Myosin Va overexpression results are consistent with these. CONCLUSIONS: SQW ameliorates overactive bladder and regulate neurotransmission via regulating Myosin Va mRNA and protein expression.
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Diabetes Mellitus Experimental , Vejiga Urinaria , Animales , Diabetes Mellitus Experimental/metabolismo , Contracción Muscular , Miosinas/metabolismo , Miosinas/farmacología , Ratas , Estreptozocina/farmacología , Transmisión Sináptica , UrodinámicaRESUMEN
OBJECTIVES: Probiotics are gaining interest as alternative options for antibiotic or antiinflammatory drugs. Probiotics can affect the health of the host through metabolites and competitive inhibition adhesion of pathogenic microorganisms. Koumiss is an important part of the diet of Asian nomads, and is rich in a broad array of probiotics that can benefit the body. Mongolians have developed koumiss therapy to assist in the treatment of various diseases. In the present study, we investigate the beneficial effect of Lactobacillus paracasei, a strain isolated from koumiss, on a mouse model of diarrhea induced by Escherichia coli O8 (E. coli O8). METHODS: Probiotics were isolated from Mongolian koumiss. The resistance of probiotics against acid, bile salts, gastric juice, and intestinal juice was evaluated. The mouse model of diarrhea was established by the intragastric administration of E. coli O8 after NaHCO3 treatment. L. paracasei was intragastrically administered before or after E. coli O8 exposure in mice. The plasma levels of diamine oxidase and zounlin were quantified using an enzyme-linked immunosorbent assay, and the integrity of the intestinal barrier and goblet cells of mice with diarrhea were observed using hematoxylin and eosin and Alcian blue periodic acid-Schiff staining. The expression of tight junction (TJ) proteins was detected by immunohistochemistry and Western blot. RESULTS: A total of five lactic acid bacteria and two yeast strains were isolated from koumiss, and L. paracasei was screened for animal experiments. Experimental results showed that L. paracasei could reduce the increase in diamine oxidase and zonulin caused by E. coli (P < 0.05); increase goblet cells and the expression of TJ proteins ZO-1, occludin, and claudin-1 (P < 0.05); increase the expression of mucin 2, oligomeric mucus/gel-forming (P < 0.05) protein; and reduce the level of inhibitor kappa B-alpha and myosin light-chain kinase. CONCLUSIONS: L. paracasei reduced the intestinal permeability, induced the expression of mucin 2, oligomeric mucus/gel-forming protein, and increased the number of goblet cells in mice by the upregulation of the expression of TJ proteins via the nuclear factor kappa B cells-myosin light-chain kinase signaling pathway.
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Amina Oxidasa (conteniendo Cobre) , Kumis , Lacticaseibacillus paracasei , Probióticos , Animales , Células CACO-2 , Diarrea/tratamiento farmacológico , Escherichia coli , Humanos , Mucosa Intestinal/metabolismo , Ratones , Mucina 2/metabolismo , Mucina 2/farmacología , Miosinas/metabolismo , Miosinas/farmacología , Probióticos/uso terapéutico , Proteínas de Uniones Estrechas , Uniones EstrechasRESUMEN
We tested the hypothesis that isoform shifts in sarcomeres of the immature heart modify the effect of cardiac myosin-directed sarcomere inhibitors and activators. Omecamtiv mecarbil (OM) activates tension and is in clinical trials for the treatment of adult acute and chronic heart failure. Mavacamten (Mava) inhibits tension and is in clinical trials to relieve hypercontractility and outflow obstruction in advanced genetic hypertrophic cardiomyopathy (HCM), which is often linked to mutations in sarcomeric proteins. To address the effect of these agents in developing sarcomeres, we isolated heart fiber bundles, extracted membranes with Triton X-100, and measured tension developed over a range of Ca2+ concentrations with and without OM or Mava treatment. We made measurements in fiber bundles from hearts of adult nontransgenic (NTG) controls expressing cardiac troponin I (cTnI), and from hearts of transgenic (TG-ssTnI) mice expressing the fetal/neonatal form, slow skeletal troponin I (ssTnI). We also compared fibers from 7- and 14-day-old NTG mice expressing ssTnI and cTnI. These studies were repeated with 7- and 14-day-old transgenic mice (TG-cTnT-R92Q) expressing a mutant form of cardiac troponin T (cTnT) linked to HCM. OM increased Ca2+-sensitivity and decreased cooperative activation in both ssTnI- and cTnI-regulated myofilaments with a similar effect: reducing submaximal tension in immature and mature myofilaments. Although Mava decreased tension similarly in cTnI- and ssTnI-regulated myofilaments controlled either by cTnT or cTnT-R92Q, its effect involved a depressed Ca2+-sensitivity in the mature cTnT-R92 myofilaments. Our data demonstrate an influence of myosin and thin-filament associated proteins on the actions of myosin-directed agents such as OM and Mava. SIGNIFICANCE STATEMENT: The effects of myosin-targeted activators and inhibitors on Ca2+-activated tension in developing cardiac sarcomeres presented here provide novel, ex vivo evidence as to their actions in early-stage cardiac disorders. These studies advance understanding of the molecular mechanisms of these agents, which are important in preclinical studies employing sarcomere Ca2+-response as a screening approach. The data also inform the use of commonly immature cardiac myocytes generated from human-inducible pluripotent stem cells in screening for sarcomere activators and inhibitors.
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Miofibrillas , Sarcómeros , Animales , Calcio/metabolismo , Ratones , Ratones Transgénicos , Contracción Miocárdica , Miocardio/metabolismo , Miocitos Cardíacos , Miofibrillas/metabolismo , Miosinas/metabolismo , Miosinas/farmacología , Troponina I/genética , Troponina I/metabolismo , Troponina I/farmacologíaRESUMEN
This experiment proposed to investigate the efficiency of omega 3 fatty acids from fish that improves autoimmune against myocarditis in the rat. Fish oil was extracted from fresh Tuna fish and performed FAME analysis and mice bioassay. The autoimmune myocarditis was induced by subcutaneous injection of porcine cardiac myosin (PCM) into the footpads of rats on the first and seventh day. Rats were dissected on the 21st day to analyze the histopathological, hemodynamic, echocardiographic factors, and immunohistochemistry expressions. In the study, 73.90% of total fatty acids were recorded. Histological analysis revealed that omega 3 fatty acids administrated groups showed tremendous development in the multifocal myocardia hyaline degeneration and necrosis with inflammatory changes. Moreover, omega 3 fatty acids inhabited the expressions of inflammatory cells (CD4, CD8 and CD11b) and suppressed the level of NF-κB. The echocardiographic factors such as heartbeat, SBP, DBP, levels of LVDs, LVDd, LVPW percentage of LVFS, EF, expression levels of inflammatory cytokines (TNF, IL-1ß, IFN-ɤ, IL-2, and IL-6) also significantly suppressed by omega 3 fatty acids. Hence, the present study proved that consuming fatty acid-enriched fish might be a successful therapy for improving the inflammatory profile, regenerates the heart tissues, and controlled the production of inflammatory cells.
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Autoinmunidad/efectos de los fármacos , Cardiotónicos/farmacología , Ácidos Grasos Omega-3/farmacología , Aceites de Pescado/química , Miocarditis/etiología , Miosinas/farmacología , Animales , Masculino , Modelos Animales , Ratas , Ratas Endogámicas Lew , PorcinosRESUMEN
Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine-binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by â¼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor V/efectos de los fármacos , Factor Xa/efectos de los fármacos , Músculo Esquelético/química , Miocardio/química , Miosinas/farmacología , Fosfolípidos/farmacología , Animales , Antígenos de Superficie/farmacología , Miosinas Cardíacas/aislamiento & purificación , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/farmacología , Bovinos , Contaminación de Medicamentos , Factor VIIa/metabolismo , Factor Xa/metabolismo , Humanos , Lipoproteínas/farmacología , Proteínas de la Leche/farmacología , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Fosfolipasas A2/farmacología , Conejos , Tromboplastina/farmacologíaRESUMEN
By using skinned-rabbit skeletal muscle fibers, the time courses of changes of thin filament-based x-ray reflections were followed at a 3.4-ms time resolution during thin-filament activation. To discriminate between the effects of calcium binding and myosin binding on thin-filament activity, measurements were performed after caged-calcium photolysis in fibers with full-filament or no-filament overlap, or during force recovery after a quick release. All three reflections examined, i.e., the second actin layer line (second ALL, reporting the tropomyosin movement), the sixth ALL (reporting actin structural change), and the meridional troponin reflections, exhibited calcium-induced and myosin-induced components, but their rate constants and polarities were different. Generally, calcium-induced components exhibited fast rate constants (>100 s(-1)). The myosin-induced components of the second ALL had a rate constant similar to that of the force (7-10 s(-1)), but that of the sixth ALL was apparently faster. The myosin-induced component of troponin reflection was the only one with negative polarity, and was too slow to be analyzed with this protocol. The results suggest that the three regulation-related proteins change their structures with different rate constants, and the significance of these findings is discussed in the context of a cooperative thin-filament activation mechanism.
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Citoesqueleto de Actina/metabolismo , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Difracción de Rayos X , Citoesqueleto de Actina/efectos de los fármacos , Animales , Fenómenos Biomecánicos , Calcio/metabolismo , Calcio/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Relajación Muscular , Miosinas/metabolismo , Miosinas/farmacología , Fotólisis , Conejos , Factores de TiempoRESUMEN
To explore the therapeutic effects of adenovirus vector mediated transfer of the ICOSIg gene on immuno-inflammation-mediated cardiac remodeling in an experimental autoimmune myocarditis (EAM) model, pAdeno-ICOSIg was constructed and transfected into HEK 293 cells to produce the ICOSIg adenovirus. Ad-CMV-GFP was used as a control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. The immunized rats were divided into two groups. The inducible co-stimulatory molecule (ICOS) group received the adenovirus containing ICOSIg on day 14; the green fluorescent protein (GFP) group received the adenovirus containing GFP as the control adenovirus and 15 normal rats (Control group) consisted of the normal controls that were not immunized. On day 28, all rats were euthanized after echocardiography and histopathologically examined for cardiac fibrosis. Western blotting was performed to detect ICOS, ICOS ligand (ICOSL), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and real-time RT-PCR was performed to detect B7-1, B7-2 and interleukin (IL)-17 expression. ELISPOT was applied to detect Th1 and Th2 cytokine production. Collagen concentration and collagen cross-linking were determined as markers of cardiac fibrosis. It was found that blockade with ICOSIg exerted antifibrotic effects on cardiac remodeling in EAM. On day 28, cardiac function and inflammatory myocardial fibrosis improved significantly in the ICOS group compared to the GFP group. The expression of ICOS, the ICOSL, B7-1 and IL-17 was statistically significantly lower in the ICOS and Control groups compared to the GFP group. ICOSIg significantly augmented Th2 cytokine production and diminished Th1 and Th17 cytokine production. This blockade of the ICOS co-stimulatory pathway with ICOSIg alleviated autoimmune inflammation-mediated cardiac remodeling and improved cardiac function. Regulation of the Th1/Th2/Th17 balance may be one of the underlying mechanisms responsible for this effect.
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Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/metabolismo , Enfermedades Autoinmunes/terapia , Terapia Genética , Miocarditis/terapia , Remodelación Ventricular , Adenoviridae/genética , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/fisiopatología , Colágeno/análisis , Citocinas/sangre , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos , Pruebas de Función Cardíaca , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Miocarditis/inmunología , Miocarditis/patología , Miocarditis/fisiopatología , Miocardio/inmunología , Miocardio/metabolismo , Miocardio/patología , Miosinas/farmacología , Ratas , Ratas Endogámicas Lew , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
AIM: To investigate whether myosin activated-T lymphocytes induce myocardial autoimmune response. METHODS: Dendritic cells and T lymphocytes were isolated and purified from spleens of Lewis rats. After cocultured in the absence or presence of cardiac myosin (20 mug/mL), the sensitized or unsensitized T lymphocytes were transferred into the naive syngeneic Lewis rats by a single tail vein infusion, respectively. The recipient rats were killed on the third day, at the first and fourth week and then the histopathological changes of the rats were observed. RESULTS: T lymphocyte infiltration was observed on the third day. It reached the peak with occasional myocardium necrosis at the first weekend. T cell infiltration distinctly lessened at the fourth weekend. No T lymphocyte infiltration was found in kidney, liver, lung, and brain. T lymphocyte infiltration wad not detected in the heart of the transferred unsensitized T lymphocytes of the rats. CONCLUSION: Myocardial autoimmune response can be induced by myosin activated T lymphocytes.
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Autoinmunidad/efectos de los fármacos , Autoinmunidad/inmunología , Miocardio/inmunología , Miosinas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Animales , Encéfalo/inmunología , Células Dendríticas/inmunología , Femenino , Citometría de Flujo , Riñón/inmunología , Hígado/inmunología , Pulmón/inmunología , Masculino , Miocardio/patología , Necrosis/inmunología , RatasRESUMEN
Malignant pleural mesothelioma (MPM) is closely related to exposure to asbestos, and a rapid increase in the number of MPM patients is therefore estimated to occur from 2010 to 2040 in Japan. Because MPM is refractory to conventional chemotherapy and radiotherapy, the prognosis of MPM patients is extremely poor. MYO18B, a novel member of the myosin family, is a tumor suppressor gene isolated from a homozygously deleted region at 22q12.1 in a lung cancer cell line. The inactivation of the MYO18B gene plays an important role in several malignant diseases. However, the role of MYO18B in the progression of MPM is still unknown. Six different human MPM cell lines were used in this study. Western blot revealed that none of the cell lines expressed a detectable level of MYO18B protein. One of the MPM cell lines, EHMES-10, was transfected with the MYO18B gene. We found that a restored expression of the MYO18B protein in EHMES-10 cells resulted in the inhibition of their anchorage-independent growth and motility in vitro. In addition, it also inhibited their ectopic (subcutaneous space) and orthotopic (thoracic cavity) growth in SCID mice, in association with an increased degree of cell apoptosis. Furthermore, it also suppressed the production of bloody pleural effusion after orthotopic injection. These findings suggest that the restored expression of MYO18B may be a useful therapeutic strategy for the treatment of locally advanced MPM in humans.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Miosinas/genética , Miosinas/farmacología , Derrame Pleural/genética , Neoplasias Pleurales/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/farmacología , Animales , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Mesotelioma/tratamiento farmacológico , Mesotelioma/metabolismo , Ratones , Ratones SCID , Miosinas/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Derrame Pleural/metabolismo , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.
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Óptica y Fotónica , Orgánulos/fisiología , Fenómenos Fisiológicos Celulares , ADN , Humanos , Cinesinas/farmacología , Miosinas/farmacología , VirusRESUMEN
Experimental autoimmune myositis (EAM) is a rodent model for human inflammatory muscle disease (IMD). It can be induced by immunization of rodents with skeletal muscle homogenate and adjuvant. The specific myositogenic autoantigen has not been clearly identified although some evidence points to skeletal muscle myosin. In this report we strengthen this evidence, showing that Lewis rats immunized with purified skeletal muscle myosin develop EAM with the same pattern and severity as EAM induced by whole rabbit skeletal muscle homogenate (WRM). Multiple inflammatory lesions are detected histopathologically in the biceps, quadriceps, and gastrocnemius muscles. Myosin-reactive T cells from animals immunized either with myosin or with WRM have similar patterns of antigen-induced proliferation. The results show that myosin, a component of skeletal muscle, is at least one autoantigen in EAM.
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Autoantígenos/inmunología , Músculo Esquelético/inmunología , Miosinas/inmunología , Enfermedad Autoinmune Experimental del Sistema Nervioso/inmunología , Polimiositis/inmunología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Adyuvante de Freund/inmunología , Adyuvante de Freund/farmacología , Inmunización , Activación de Linfocitos , Músculo Esquelético/patología , Miosinas/farmacología , Enfermedad Autoinmune Experimental del Sistema Nervioso/patología , Polimiositis/patología , Conejos , Ratas , Ratas Endogámicas Lew , Linfocitos T/inmunologíaRESUMEN
Alteration of troponin T (TnT) isoform expression has been reported in human and animal models of myocardial failure. The two adult beef cardiac TnT isoforms (TnT(3) and TnT(4)) were isolated for comparative functional analysis. Thin filaments were reconstituted containing pure populations of the isoforms. The in vitro motility assay was used to directly compare the effect of the two TnT isoforms on force and unloaded shortening as a function of free calcium. We found no significant differences between the two isoforms in terms of calcium sensitivity, cooperativity, or maximal activation (velocity and force) as assessed in a fully calcium-regulated system. Activation by myosin strong binding was similar for thin filaments containing either of the two TnT isoforms. Whereas maximally activated velocity and cooperativity was depressed at pH 6.5, no difference between thin filaments containing the two isoforms was detected. From the small magnitude of the TnT isoform shifts detected in myocardial failure and the lack of significant mechanical effect detected in the motility assay, variable TnT isoform expression is unlikely to be any functional significance in heart failure.
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Contracción Muscular/efectos de los fármacos , Isoformas de Proteínas/farmacología , Troponina T/farmacología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Actinas/aislamiento & purificación , Actinas/metabolismo , Actinas/farmacología , Animales , Fenómenos Biomecánicos , Calcio/farmacología , Bovinos , Pollos , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Contracción Isométrica , Músculo Esquelético/química , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Miosinas/farmacología , Conejos , Troponina T/aislamiento & purificaciónRESUMEN
Combretastatin A-4-phosphate (CA-4-P) is a tubulin-binding compound currently in clinical trial as a tumor vascular-targeting agent. In endothelial cells, CA-4-P is known to cause microtubule depolymerization, but little is known about its subsequent effects on cell morphology and function. Here, we demonstrate that within minutes of endothelial cell exposure to CA-4-P, myosin light chain (MLC) was phosphorylated, leading to actinomyosin contractility, assembly of actin stress fibers, and formation of focal adhesions. These cytoskeletal alterations appeared to be a consequence of Rho activation, as they were abolished by either the Rho inhibitor C3 exoenzyme or Rho-kinase inhibitor Y-27632. In response to CA-4-P, some cells rapidly assumed a blebbing morphology in which F-actin accumulated around surface blebs, stress fibers misassembled into a spherical network surrounding the cytoplasm, and focal adhesions appeared malformed. Blebbing was associated with decreased cell viability and could be inhibited by Rho/Rho-kinase inhibitors or by blocking the CA-4-P-mediated activation of stress-activated protein kinase-2/p38. The extracellular-regulated kinases 1 and 2 (ERK-1/2) were shown to protect against blebbing since blebbing was attenuated on ERK-1/2 stimulation and was up-regulated by specific inhibition of ERK-1/2 activation. The use of MLC kinase (MLCK) and myosin adenosine triphosphatase inhibitors led us to propose a role for MLCK and myosin activity independent of MLC phosphorylation in regulating the blebbing process. CA-4-P-mediated contractility and blebbing were associated with a Rho-dependent increase in monolayer permeability to dextrans, suggesting that such functional changes may be important in the rapid response of the tumor endothelium to CA-4-P in vivo.
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Actinas/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacología , Citoesqueleto/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/ultraestructura , Estilbenos/farmacología , Actinas/metabolismo , Proteínas de Fase Aguda/efectos de los fármacos , Proteínas de Fase Aguda/farmacología , Proteínas de Fase Aguda/fisiología , Permeabilidad Capilar/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Endotelio Vascular/citología , Adhesiones Focales/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/farmacología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Quinasa de Cadena Ligera de Miosina/farmacología , Quinasa de Cadena Ligera de Miosina/fisiología , Miosinas/farmacología , Miosinas/fisiología , Necrosis , Fibras de Estrés/efectos de los fármacosRESUMEN
Experimental autoimmune myositis (EAM) is a good model of human inflammatory myopathy. We induced EAM in SJL/J mice by injection with myosin and treated inflammatory changes with FK506. The mice developed inflammatory changes after the fifth myosin injection. After treatment with FK506, inflammation was suppressed and central nuclei of the muscle fibers increased. These findings indicate that FK506 is effective in the treatment of EAM. The data suggests that FK506 inhibits interaction with calcineurin. Intercellular adhesion molecule-1 (ICAM-1) positive cells were present in the inflammatory and non-inflammatory areas of EAM. The FK506-treated group stained more weakly for ICAM-1 than the untreated EAM group.
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Inmunosupresores/farmacología , Músculo Esquelético/efectos de los fármacos , Miositis/tratamiento farmacológico , Enfermedad Autoinmune Experimental del Sistema Nervioso/tratamiento farmacológico , Tacrolimus/farmacología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/inmunología , Núcleo Celular/patología , Femenino , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Miosinas/inmunología , Miosinas/farmacología , Miositis/inmunología , Miositis/metabolismo , Enfermedad Autoinmune Experimental del Sistema Nervioso/inmunología , Enfermedad Autoinmune Experimental del Sistema Nervioso/metabolismo , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Resultado del TratamientoRESUMEN
Growth cones are required for the forward advancement and navigation of growing axons. Modulation of growth cone shape and reorientation of the neurite are responsible for the change of outgrowth direction that underlies navigation. Change of shape involves the reordering of the cytoskeleton. Reorientation of the neurite requires the generation of tension, which is supplied by the ability of the growth cone to crawl on a substrate. The specific molecular mechanisms responsible for these activities are unknown but are thought to involve actomyosin-generated force combined with linkage to the cell surface receptors that are responsible for adhesion (Heidemann and Buxbaum, 1998). To test whether myosin IIB is responsible for the force generation, we quantified shape dynamics and filopodial-mediated traction force in growth cones from myosin IIB knock-out (KO) mice and compared them with neurons from normal littermates. Growth cones from the KO mice spread less, showed alterations in shape dynamics and actin organization, and had reduced filopodial-mediated traction force. Although peak traction forces produced by filopodia of KO cones were decreased significantly, KO filopodia occasionally developed forces equivalent to those in the wild type. This indicates that other myosins participate in filopodial-dependent traction force. Therefore, myosin IIB is necessary for normal growth cone spreading and the modulation of shape and traction force but acts in combination with other myosins for some or all of these activities. These activities are essential for growth cone forward advancement, which is necessary for outgrowth. Thus outgrowth is slowed, but not eliminated, in neurons from the myosin IIB KO mice.
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Conos de Crecimiento/metabolismo , Miosinas/metabolismo , Resinas Acrílicas/metabolismo , Resinas Acrílicas/farmacología , Actinas/metabolismo , Actinas/ultraestructura , Animales , Células Cultivadas , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/ultraestructura , Ratones , Ratones Noqueados , Microscopía Fluorescente , Microesferas , Cadenas Pesadas de Miosina/deficiencia , Cadenas Pesadas de Miosina/genética , Miosinas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Miosina Tipo IIB no Muscular , Fenotipo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Seudópodos/ultraestructura , Estrés Mecánico , Ganglio Cervical Superior , Factores de TiempoRESUMEN
Rheumatic heart disease is an autoimmune sequela of group A streptococcal infection. Previous studies have established that streptococcal M protein is structurally and immunologically similar to cardiac myosin, a well-known mediator of inflammatory heart disease. In this study, we investigated the hypothesis that streptococcal M protein could produce inflammatory valvular heart lesions similar to those seen in rheumatic fever (RF). Fifty percent (3 of 6) of Lewis rats immunized with recombinant type 6 streptococcal M protein (rM6) developed valvulitis as well as focal lesions of myocarditis. Valvular lesions initiated at the valve surface endothelium spread into the valve. Anitschkow cells and verruca-like lesions were present. T cells from rM6-immunized rats proliferated in the presence of purified cardiac myosin, but not skeletal myosin. A T-cell line produced from rM6-treated rats proliferated in the presence of cardiac myosin and rM6 protein. The study demonstrates that the Lewis rat is a model of valvular heart disease and that streptococcal M protein can induce an autoimmune cell-mediated immune attack on the heart valve in an animal model. The data support the hypothesis that a bacterial antigen can break immune tolerance in vivo, an important concept in autoimmunity.
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Antígenos Bacterianos , Enfermedades Autoinmunes/fisiopatología , Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/inmunología , Enfermedades de las Válvulas Cardíacas/fisiopatología , Animales , Enfermedades Autoinmunes/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Línea Celular , Modelos Animales de Enfermedad , Enfermedades de las Válvulas Cardíacas/inmunología , Humanos , Inmunización , Activación de Linfocitos , Válvula Mitral/patología , Miocarditis/inmunología , Miocarditis/fisiopatología , Miosinas/farmacología , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
We have studied the effects of three compounds on surface oscillations of human red blood cell ghosts: the P-ATPase inhibitor, suramin; the fluorescent dye of a similar structure, 1,8-anilinonaphthalene sulfonate (ANS); and subfragment 1 of skeletal muscle myosin (S1). It has been found that suramin (10 microM), ANS (100 microM) and S1 (2 mg/ml) suppress the surface oscillations reversibly. The shape of the ghosts remains unchanged. We have also found that suramin and ANS inhibit the ghosts' non-transport (presumably, F-actin-associated) ATPase. The results of the present study suggest the important role of actin ATPase in the generation of cell surface oscillations. The effect of S1, the protein which increases the torsional, but not the bending, rigidity of F-actin upon binding to filaments, favours the possibility that just the torsional dynamics of actin protofilaments leads to the observed oscillations of the ghosts' surface.