RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The incidence of membranous nephropathy (MN) continues to rise globally. Shenqi granule (SQ), composed of thirteen Chinese medicinal herbs, has clinical efficacy in the treatment of MN and has been used in China for decades. However, the mechanism behind this effect remains unclear. AIM OF THE STUDY: In this study, we documented the effects of SQ on cultured mouse podocytes (MPC5) cytoskeletal proteins (CD2AP, α-actinin4) and autophagic activity, and identified the mechanism underlying the ameliorating effects of SQ on MN. MATERIALS AND METHODS: The main components of SQ was analysed using High-performance liquid chromatography (HPLC). We induced MPC5 cells with puromycin aminonucleoside (PAN) as a model of MN-like disease. Cyclosporine A (CsA) was used as a positive control drug. MPC5 cells viability was analysed using CCK-8 assays to select the PAN dose and SQ dose. CD2AP and α-actinin4 mRNA expression was examined by RT-PCR, CD2AP and α-actinin4 protein expression as well as autophagic activity (LC3, Beclin1) was examined by Western blot in MPC5 cells, and the mechanism of action of SQ granule was assessed by Western blot to detect the protein expression at the phosphorylation level of PI3K/AKT/mTOR pathway. RESULTS: In PAN-induced MPC5 cells, mRNA and protein expression of α-actinin-4 and CD2AP were significantly reduced, and SQ granule was able to alleviate this manifestation. In contrast to the inhibition of LC3 and Beclin1 expression in the PAN model, SQ granule was able to activate cellular autophagic activity. In addition to this, our study revealed that PAN could activate the mTOR/ULK1 pathway, resulting in a significant increase in p-mTOR and p-ULK1 protein expression, while the SQ group was able to significantly inhibit the phosphorylation level of this pathway. CONCLUSIONS: SQ granule attenuated PAN-induced MPC5 cell damage similar to MN. The mechanism may be to upregulate the expression of α-actinin-4 and CD2AP and activate autophagy activity, which may be achieved by inhibiting the phosphorylation level of mTOR/ULK1.
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Actinina , Podocitos , Animales , Ratones , Actinina/metabolismo , Actinina/farmacología , Autofagia , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Background: Regular, especially sustained exercise plays an important role in the prevention and treatment of multiple chronic diseases. Some of the underlying molecular and cellular mechanisms behind the adaptive response to physical activity are still unclear, but recent findings suggest a possible role of epigenetic mechanisms, especially miRNAs, in the progression and management of exercise-related changes. Due to the combination of the analysis of epigenetic biomarkers (miRNAs), the intake of food and supplements, and genetic dispositions, a "fitness score" was evaluated to assess the individual response to nutrition, exercise, and metabolic influence. Methods: In response to a 12-week sports intervention, we analyzed genetic and epigenetic biomarkers in capillary blood from 61 sedentary, healthy participants (66.1% females, 33.9% males, mean age 33 years), including Line-1 methylation, three SNPs, and ten miRNAs using HRM and qPCR analysis. These biomarkers were also analyzed in a healthy, age- and sex-matched control group (n, 20) without intervention. Food frequency intake, including dietary supplement intake, and general health questionnaires were surveyed under the supervision of trained staff. Results: Exercise training decreased the expression of miR-20a-5p, -22-5p, and -505-3p (p < 0.02) and improved the "fitness score," which estimates eight different lifestyle factors to assess, nutrition, inflammation, cardiovascular fitness, injury risk, regeneration, muscle and hydration status, as well as stress level. In addition, we were able to determine correlations between individual miRNAs, miR-20a-5p, -22-5p, and -101-3p (p < 0.04), and the genetic predisposition for endurance and/or strength and obesity risk (ACE, ACTN3, and FTO), as well as between miRNAs and the body composition (p < 0.05). MiR-19b-3p and -101-3p correlated with the intake of B vitamins. Further, miR-19b-3p correlated with magnesium and miR-378a-3p with iron intake (p < 0.05). Conclusions: In summary, our results indicate that a combined analysis of several biomarkers (miRNAs) can provide information about an individual's training adaptions/fitness, body composition, nutritional needs, and possible recovery. In contrast to most studies using muscle biopsies, we were able to show that these biomarkers can also be measured using a minimally invasive method.
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MicroARNs , Actinina/metabolismo , Adulto , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Biomarcadores , Composición Corporal , Dieta , Ejercicio Físico/fisiología , Femenino , Humanos , Masculino , MicroARNs/genéticaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Huangqi Guizhi Wuwu Decoction(HQGZWWD) is a Traditional Chinese Medicine formula from Synopsis of Golden Chamber used to treat blood arthralgia. According to the principle that the same treatment can be used for different diseases, HQGZWWD has proven effective for IgA nephropathy (IgAN) associated with spleen and kidney yang deficiency. AIM OF THE STUDY: In this study, we investigated the mechanism by which HQGZWWD alleviates proteinuria and protects renal function in rats with IgAN by regulating the AT1R/Nephrin/c-Abl pathway. METHODS: Rats were randomly divided into six groups: control, IgAN model, IgAN model treated with low-dose HQGZWWD, IgAN model treated with medium-dose HQGZWWD, IgAN model treated with high-dose HQGZWWD, and IgAN model treated with valsartan. IgAN was induced using bovine γ-globulin. We evaluated the mediating effects of HQGZWWD on podocyte cytoskeletal proteins, the AT1R/Nephrin/c-Abl pathway, upstream tumor necrosis factor-α (TNF-α), and TNF-α receptor-1 (TNFR1). RESULTS: The IgAN rats displayed proteinuria, IgA deposition in the mesangial region, and podocyte cytoskeletal protein damage. The expression of TNF-α, TNFR1, AT1R, and c-Abl was increased in the IgAN rat kidney, whereas the expression of nephrin, podocin, ACTN4, and phosphorylated nephrin (p-nephrin) was reduced. HQGZWWD treatment significantly alleviated podocyte cytoskeletal protein damage in the IgAN rats, upregulated the expression of nephrin, podocin, and ACTN4, and the colocalized expression of F-actin and nephrin. This study demonstrates that HQGZWWD attenuates podocyte cytoskeletal protein damage by regulating the AT1R-nephrin- c-Abl pathway, upregulating the expression of p-nephrin, and downregulating the expression of AT1R and c-Abl. CONCLUSIONS: These results indicate that HQGZWWD attenuates podocyte cytoskeletal protein damage in IgAN rats by regulating the AT1R/Nephrin/c-Abl pathway, providing a potential therapeutic approach for IgAN.
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Proteínas del Citoesqueleto/metabolismo , Medicamentos Herbarios Chinos/farmacología , Glomerulonefritis por IGA/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Sustancias Protectoras/farmacología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/uso terapéutico , Glomerulonefritis por IGA/metabolismo , Glomerulonefritis por IGA/patología , Glomerulonefritis por IGA/fisiopatología , Inmunoglobulina A/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/genética , Podocitos/efectos de los fármacos , Sustancias Protectoras/química , Sustancias Protectoras/uso terapéutico , Proteinuria/metabolismo , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Traditional Chinese medicine (TCM) preparations have significant effects on some refractory diseases; however, these compositions are complex and their mechanisms are unknown. Identification of the active components in these preparations is essential. The mortality rate for heart failure (HF) has been increasing in recent years, and myocardial dysfunction (MD) has been proved to be the pathological basis of HF. Yixinshu Capsule (YXSC) is a multi-component oral drug with therapeutic effects on HF. However, the key active components are still unclear. In this study, YXSC intestinal absorption liquid (IAL) was used and 62 compounds were identified by an analytical chemistry approach. Then, a compound - target - function network was established with a bioinformatics analysis tool. Finally, a cell model of MD on human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) was used to verify the therapeutic effects of the active components of YXSC. Schisandrin A (Sch A) and schisandrin B (Sch B) were demonstrated to be the active components of YXSC by attenuating endothelin-1 (ET-1)-induced contraction dysfunction, brain natriuretic peptide (BNP) content elevation, and the morphological changes of hiPS-CMs. For the first time, our data illustrate the potent protective effects of Sch A and Sch B on ET-1-induced dysfunctional hiPS-CMs and revealed their effective targets and pathways. The integrative approach used in our study was applied to identify active components in TCM preparations and excavate the possible mechanisms.
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Cardiotónicos/farmacología , Ciclooctanos/farmacología , Medicamentos Herbarios Chinos/química , Antagonistas de los Receptores de Endotelina/farmacología , Lignanos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Compuestos Policíclicos/farmacología , Actinina/antagonistas & inhibidores , Actinina/genética , Actinina/metabolismo , Animales , Bosentán , Cardiotónicos/química , Cardiotónicos/aislamiento & purificación , Diferenciación Celular , Línea Celular , Ciclooctanos/química , Ciclooctanos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/aislamiento & purificación , Endotelina-1/antagonistas & inhibidores , Endotelina-1/farmacología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Lignanos/química , Lignanos/aislamiento & purificación , Masculino , Medicina Tradicional China , Redes y Vías Metabólicas/efectos de los fármacos , Metaboloma , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/antagonistas & inhibidores , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Compuestos Policíclicos/química , Compuestos Policíclicos/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacología , Troponina T/antagonistas & inhibidores , Troponina T/genética , Troponina T/metabolismoRESUMEN
Restoration of blood flow to the ischemic myocardium is imperative to avoid demise of cardiomyocytes, but is paradoxically associated with irreversible damage to cardiac tissues due to the excessive generation of reactive oxygen species (ROS). We have previously reported that saffron, a natural antioxidant, attenuated ischemia-reperfusion (IR) injuries in vitro; however, its role in a meaningful cardiac recovery remains unknown. Here, we show that saffron supplement (oral administration for 6 weeks) reduced myocardial damage and restored cardiac function in an IR model of rabbit hearts. This was evidenced by improved left ventricle pressure, heart rate and coronary flow, and left ventricle end diastolic pressure (LVEDP) in IR hearts (isolated from rabbits pre-exposed to saffron (S/IR)). Electrophysiological recordings revealed a significant decline in both premature ventricle contraction and ventricle tachycardia/fibrillation in S/IR compared to IR hearts. This was paralleled by increased expression of the contractile proteins α-actinin and Troponin C in the myocardium of S/IR hearts. Histological examination combined to biochemical analysis indicated that hearts pre-exposed to saffron exhibited reduced infarct size, lower lipid peroxidation, with increased glutathione peroxidase activity, and oxidation of nitro blue tetrazolium (by reactive oxygen species). Furthermore, in contrast with IR hearts, saffron pretreatment induced restoration of the phosphorylation level of the survival proteins Akt and 4EBP1 and reduced activity of p38. Collectively, our data demonstrate that the natural antioxidant saffron plays a pivotal role in halting IR-associated cardiac injuries and emerges as a novel preventive tool for ischemic heart disease.
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Cardiotónicos/farmacología , Crocus/química , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Actinina/genética , Actinina/metabolismo , Administración Oral , Animales , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Técnicas de Cultivo de Órganos , Estrés Oxidativo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Conejos , Especies Reactivas de Oxígeno/metabolismo , Troponina C/genética , Troponina C/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND/AIMS: Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA. METHODS: The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE, 50 µmol/L, 48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 µg/ml, 48 h). The cell surface area was determined by immunofluorescent staining for α-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and ß-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio. The miR-199a-5p expression level was quantified in PE-treated cardiomyocytes and heart samples from acute myocardial infarction (AMI) mouse model. MiR-199a-5p overexpression was used to determine its role in the anti-hypertrophic effect of QL on cardiomyocytes. RESULTS: PE induced obvious enlargement of cell surface in cardiomyocytes, paralleling with increased ANP, BNP, and MYH7 mRNA levels and elevated protein/DNA ratio. All these changes were reversed by the treatment with QL. Meanwhile, miR-199a-5p was increased in AMI mouse heart tissues. Of note, the increase of miR-199a-5p in PE-treated cardiomyocytes was reversed by the treatment with QL. Moreover, overexpression of miR-199a-5p abolished the anti-hypertrophic effect of QL on cardiomyocytes. CONCLUSION: QL prevents PE-induced cardiac hypertrophy. MiR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes.
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Regulación hacia Abajo/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , MicroARNs/metabolismo , Actinina/metabolismo , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/uso terapéutico , Medicina Tradicional China , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Oligonucleótidos Antisentido/metabolismo , Fenilefrina/toxicidad , Ratas , Ratas Sprague-DawleyRESUMEN
Aristolochic acid I (AA-I) is a strong nephrotoxin, carcinogen, and mutagen found in plants such as the Aristolochia species. The mechanisms underlying AA-I toxicity in the kidneys are poorly understood. In this study, we aimed to gain insight into the mechanism of AA-I nephrotoxicity by analyzing the uptake, subcellular distribution, and intracellular targets of AA-I in the human kidney cell line HK-2 using immunocytochemistry, immunoprecipitation, and LC-MS/MS. In HK-2 cells incubated with 20[Formula: see text][Formula: see text]g/mL AA-I for different periods of time (up to 12[Formula: see text]h), AA-I was detected by a specific monoclonal antibody (MAb) against AA-I, both in the cytoplasm and nuclei. Nuclear localization depended on the exposure time. A protein with the molecular weight of 100 kDa was immunoprecipitated with the anti-AA-I MAb from the AA-I-treated cell lysates and was identified by LC-MS/MS as [Formula: see text]-actinin-4 after digestion of the protein, and was confirmed by immunoblotting with a specific anti-[Formula: see text]-actinin-4 MAb. This evidence shows, for the first time, that [Formula: see text]-actinin-4 is a protein targeted by AA-I in kidney cells. Our findings strongly suggest an association between [Formula: see text]-actinin-4 and AA-I nephrotoxic activity.
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Actinina/metabolismo , Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/toxicidad , Riñón/metabolismo , Células Cultivadas , Cromatografía Liquida , Humanos , Inmunohistoquímica , Inmunoprecipitación , Riñón/citología , Espectrometría de Masas en TándemRESUMEN
Herbal remedies are often used during the early stages of pregnancy, being considered 'harmless' and 'natural'. There are insufficient data regarding their potential embryotoxicity. The main components of selected herbs, including 6-gingerol from ginger, Ginkgolide A and Ginkgolide B from gingko biloba and Ginsenoside Rg1 from ginseng, have been investigated using chick embryonic heart micromass and Mouse D3 embryonic stem cells. The potential effects were evaluated via alteration in contractility, cell viability, and cell protein content. The myocytes in both systems were also demonstrated by immunocytochemistry using a specific cardiomyocyte marker (α-actinin). For 6-gingerol, Ginkgolide A, Ginkgolide B and Ginsenoside Rg1 in both methods, at moderate to high concentrations, there were alterations in the values for the endpoints. These data indicate that herbal remedies used in the first trimester of pregnancy might not be safe for fetal development.
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Células Madre Embrionarias/efectos de los fármacos , Corazón/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Preparaciones de Plantas/toxicidad , Actinina/metabolismo , Animales , Biomarcadores/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , Corazón/embriología , Corazón/fisiopatología , Ratones , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Preparaciones de Plantas/análisis , Medición de RiesgoRESUMEN
The authors compared ACTN3 R577X genotype and allele frequencies in the majority of all-time-best Spanish judo male athletes (n = 108) and 343 ethnically matched nonathletic men. No between-groups differences were found in allele (P = .077) or genotype distributions (P = .178). Thus, the R577X polymorphism was not significantly associated with the status of being an elite judo athlete, at least in the Spanish population. The contribution of genetics to sports-related phenotype traits is undeniable with some genotypes, of which ACTN3 R577X is currently the leading candidate, partly distinguishing individuals predisposed to either endurance or power sports. However, few athletic events can be categorized as purely power or endurance based. Although genetic testing (ie, for ACTN3 R577X) is already being marketed to predict sports talent and potential of young children, its usefulness is still questionable, at least in competitive judo.
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Actinina/genética , ADN/genética , Artes Marciales/fisiología , Resistencia Física/genética , Polimorfismo Genético , Deportes/fisiología , Actinina/metabolismo , Adolescente , Adulto , Anciano , Alelos , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Adulto JovenRESUMEN
Chemical approaches are widely used in directed differentiation of embryonic stem (ES) cells. In our search for novel lead compounds that could facilitate cardiomyogenesis of ES cells, we designed a two-step screening system based on P19 embryonic carcinoma and mouse ES cells. Application of this system to a quinazoline compound library including 2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines and 2,6-disubstituted 4-anilinoquinazoline led us to the discovery of compound 62, which exhibits a stable cardiomyogenic effect on both P19 and mouse ES cells at a concentration of 0.1 µM. An EGFR inhibition assay and molecular docking studies confirmed 62 as a potent EGFR inhibitor with a tyrosine kinase IC(50) value of 101 nM. However, major differences in cardiomyogenic activity were observed between iressa and 62, indicating that other molecular events are also involved in compound 62-induced cardiomyogenesis of ES cells.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/citología , Quinazolinas/química , Quinazolinas/farmacología , Actinina/metabolismo , Animales , Factor Natriurético Atrial/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Receptores ErbB/antagonistas & inhibidores , Concentración 50 Inhibidora , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas/síntesis química , Sarcómeros/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
Invasion of normal brain tissue by brain tumor cells is a major contributing factor to the recurrence and resistance of clinically diagnosed glioblastomas to therapy (surgery, chemotherapy, radiation). Here, we have assessed the efficacy of the microtubule inhibiting agent epothilone B on glioblastoma cell motility, a prerequisite cellular program of invasive glioblastomas. Using cell migration assays and immunofluorescence techniques we demonstrated that epothilone B abrogated glioblastoma cell motility as a consequence of α-actinin 4 redistristrubiton and the breakdown of cellular structures (leading edge, stress fibers) it is associated with during cell migration. Evaluation of the microtubule actin cross linking factor in glioblastoma cells also revealed epothilone B invoked changes in this cytoskeleton cross linking protein, resembling α-actinin 4 changes in response to epothilone B. We have demonstrated in this study that epothilone B antagonizes glioblastoma cell motility due to the disruption of cytoskeleton binding proteins that aide in preserving the structural organization of the cytoskeleton filamentous network. Furthermore, we provide preclincial evidence that epothilone B effects on glioblastomas are not limited to the impairment of dividing tumors cells but that it also targets migratory and invasive glioblastoma cells, suggesting that this agent has potential clinical benefit due to its ability to target divergent cellular programs in the glioblastoma tumor mass.
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Actinina/antagonistas & inhibidores , Neoplasias Encefálicas/patología , Movimiento Celular/efectos de los fármacos , Epotilonas/farmacología , Glioma/patología , Moduladores de Tubulina/farmacología , Actinina/metabolismo , Antineoplásicos/farmacología , Neoplasias Encefálicas/metabolismo , Regulación hacia Abajo , Evaluación Preclínica de Medicamentos , Glioma/metabolismo , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Multimerización de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C(2)H(2)-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers.
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Actinina/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Proteínas WT1/metabolismo , Cinética , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , ARN/metabolismo , Resonancia por Plasmón de Superficie , Factores de Transcripción/genética , Proteínas WT1/genética , Dedos de Zinc/genéticaRESUMEN
BACKGROUND: Animal studies suggest that reactive oxygen species (ROS) play an important role in the development of diabetic cardiomyopathy. HYPOTHESIS: Matrix metalloproteinase-2 (MMP-2) is activated by ROS and contributes to the acute loss of myocardial contractile function by targeting and cleaving susceptible proteins including troponin I (TnI) and alpha-actinin. METHODS: Using the streptozotocin-induced diabetic rat model, we evaluated the effect of daily in vivo administration of sodium selenate (0.3 mg/kg; DMS group), or a pure omega-3 fish oil with antioxidant vitamin E (omega-3E; 50 mg/kg; DMFA group), which has antioxidant-like effects, for 4 weeks on heart function and on several biochemical parameters related to oxidant stress and MMP-2. RESULTS: Although both treatments prevented the diabetes-induced depression in left ventricular developed pressure (LVDP) as well as the rates of changes in developed pressure (+/-dP/dt) (P<.001), the improvement in LVDP of the DMS group was greater compared to that of the DMFA group (P<.001). Moreover, these treatments reduced the diabetes-induced increase in myocardial oxidized protein sulfhydryl and nitrite concentrations (P<.001). Gelatin zymography and Western blot data indicated that the diabetes-induced changes in myocardial levels of MMP-2 and tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) and the reduction in TnI and alpha-actinin protein levels were improved in both the DMS and DMFA groups (P<.001). CONCLUSIONS: These results suggest that diabetes-induced alterations in MMP-2 and TIMP-4 contribute to myocardial contractile dysfunction by targeting TnI and alpha-actinin and that sodium selenate or omega-3E could have therapeutic benefits in diabetic cardiomyopathy.
Asunto(s)
Actinina/metabolismo , Antioxidantes/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Corazón/fisiopatología , Troponina I/metabolismo , Actinina/efectos de los fármacos , Animales , Ácidos Grasos Omega-3/uso terapéutico , Corazón/efectos de los fármacos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Contracción Miocárdica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Ácido Selénico , Compuestos de Selenio/uso terapéutico , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Troponina I/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
OBJECTIVE: To examine suppressive effects of multi-glycoside of Tripterygium wilfordii Hook. f. (GTW)on mesangial injury induced by two-injecti on of anti-Thy1. 1 monoclonal antibody(mAb) 1-22-3 in vitro. METHOD: We established the irreversible model of glomerulosclerosis with anti-Thy1. 1 mAb 1-22-3. After 42 days of oral treatment with GTW (50 mg x kg(-1) BW)and vehicle (distilled water), to observe effects of GTW on proteinuria, renal function, mesangial morphological change, and mRNA expressions of collagen type I and TGF-beta by light microscope (LM), immunofluorescence (IF), and Reverse Transcription Polymerase Chain Reaction (RT-PCR). RESULT: GTW ameliorated proteinuria (from day24 to day 42) and mesangial proliferation [total cell number, GTW group 65.67+/-3.43 vs. control group 87.02+/-2.41, P < 0.05; matrix expansion, GTW group 1.20+/-0.06 vs. control group 2.77+/-0.23, P < 0.05; alpha-smooth muscle actin(alpha-SMA) expression, GTW group 1.75+/-0.33 vs. control group 2.62+/-0.15, P < 0.05; collagen type I expression, GTW group 1.68+/-0.31 vs. control group 2.06+/-0.24, P < 0.05], moreover, significantly reduced the glomerular expression of mRNA for collagen type 1(53.5% to the control group, P < 0.05)and TGF-beta(14.7% to the control group, P < 0.05)on day 42day. CONCLUSION: GTW can not only decrease proteinuria, but also ameliorate mesangial alterations probably by the reduction of cytokines. GTW may be a promising agent for the prevention of progressive and irreversible glomerulosclerosis.
Asunto(s)
Colágeno Tipo I/biosíntesis , Mesangio Glomerular/patología , Glomerulonefritis Membranoproliferativa/metabolismo , Glicósidos/farmacología , Tripterygium , Actinina/metabolismo , Animales , Colágeno Tipo I/genética , Femenino , Mesangio Glomerular/metabolismo , Glomerulonefritis Membranoproliferativa/patología , Glicósidos/aislamiento & purificación , Plantas Medicinales/química , Proteinuria/tratamiento farmacológico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Tripterygium/químicaRESUMEN
Calpain 1 behaviour toward cytoskeletal targets was investigated using two alpha-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(-Ca2+) = 0.5 +/- 0.1 microM] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 +/- 0.01 microM]. Location of the binding structures shows that the C-terminal domain of alpha-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and alpha-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and alpha-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.
Asunto(s)
Actinina/metabolismo , Calpaína/metabolismo , Citoesqueleto/metabolismo , Actinina/química , Animales , Sitios de Unión , Calcio/metabolismo , Calcio/farmacología , Calpaína/química , Bovinos , Línea Celular , Humanos , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismoRESUMEN
Immunohistochemical staining of 5 cytoskeletal proteins (actin, alpha-actinin, gelsolin, plectin and plakoglobin) was used to investigate changes in distribution patterns of these proteins after the period of uterine receptivity for blastocyst implantation in the rat. Actin was found throughout the cytoplasm but it was concentrated along the apical plasma membrane on day 1 of pregnancy, decreased by day 6 and then increased again at day 9. Alpha-actinin and gelsolin were localized in distinctive bands along the apical plasma membrane at day 6 of pregnancy but became diffusely distributed at day 9. Plectin was localized along the apical and basal plasma membranes at day 6 but in higher amounts apically and at day 9, it was concentrated in apical and basal zones in the cells. Plakoglobin was found along the lateral and basal membranes with increased intensity along the apical third of the lateral plasma membrane from day 6 to day 9 of pregnancy. These results show that all 5 cytoskeletal proteins redistributed after the period of uterine receptivity: some exhibited a similar pattern of labelling to that found during the prereceptive state, whereas others only partially returned to the pre-receptive state. This change in distribution patterns may reflect differences in the epithelial barrier function before and after the period of receptivity.
Asunto(s)
Citoesqueleto/metabolismo , Células Epiteliales/citología , Útero/patología , Actinina/biosíntesis , Actinina/metabolismo , Actinas/biosíntesis , Actinas/metabolismo , Animales , Citoplasma/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/metabolismo , Desmoplaquinas , Implantación del Embrión , Femenino , Gelsolina/biosíntesis , Gelsolina/metabolismo , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Filamentos Intermediarios/metabolismo , Pectinas/metabolismo , Plectina , Ratas , Ratas Wistar , Factores de Tiempo , Distribución Tisular , Útero/citología , gamma CateninaRESUMEN
The yeast two-hybrid system was used to search for interaction partners of human zyxin. Screening of two different cDNA libraries, one prepared from human placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, nebulette, and alpha-actinin. The zyxin/alpha-actinin interaction was analyzed in detail. By site-directed mutagenesis, a linear motif of 6 amino acids (Phe-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role. Replacement of a single amino acid within this motif abolished binding to alpha-actinin in blot overlays as well as in living cells. On the other hand, the interaction site in alpha-actinin was mapped to a conformational determinant present in the center of the protein as demonstrated by a fragment deletion analysis. This binding site involved a tandem array of two complete spectrin-like domains. Only fragments that were able to dimerize in yeast also bound to zyxin, suggesting that dimerization of alpha-actinin is essential for zyxin binding.
Asunto(s)
Actinina/química , Actinina/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Secuencias de Aminoácidos , Aminoácidos/química , Animales , Sitios de Unión , Células COS , Células Cultivadas , Embrión de Pollo , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Dimerización , Fibroblastos/metabolismo , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Glicoproteínas , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Miocardio/metabolismo , Placenta/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Activación Transcripcional , Transfección , Técnicas del Sistema de Dos Híbridos , ZixinaRESUMEN
The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.
Asunto(s)
Calcineurina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Musculares/metabolismo , Actinina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Calcineurina/genética , Proteínas Portadoras/genética , Chlorocebus aethiops , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica , Corazón/embriología , Humanos , Ratones , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Estructura Terciaria de Proteína , Conejos , Sarcómeros/metabolismo , Factores de TiempoRESUMEN
The assembly of stable cytoskeletal structures from dynamically recycled molecules requires developmental and spatial regulation of protein interactions. In muscle, titin acts as a molecular ruler organizing the actin cytoskeleton via interactions with many sarcomeric proteins, including the crosslinking protein alpha-actinin. An interaction between the C-terminal domain of alpha-actinin and titin Z-repeat motifs targets alpha-actinin to the Z-disk. Here we investigate the cellular regulation of this interaction. alpha-actinin is a rod shaped head-to-tail homodimer. In contrast to C-terminal fragments, full-length alpha-actinin does not bind Z-repeats. We identify a 30-residue Z-repeat homologous sequence between the actin-binding and rod regions of alpha-actinin that binds the C-terminal domain with nanomolar affinity. Thus, Z-repeat binding is prevented by this 'pseudoligand' interaction between the subunits of the alpha-actinin dimer. This autoinhibition is relieved upon binding of the Z-disk lipid phosphatidylinositol-bisphosphate to the actin-binding domain. We suggest that this novel mechanism is relevant to control the site-specific interactions of alpha-actinin during sarcomere assembly and turnover. The intramolecular contacts defined here also constrain a structural model for intrasterical regulation of all alpha-actinin isoforms.
Asunto(s)
Actinina/metabolismo , Proteínas Musculares/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Unión Competitiva , Calorimetría , Células Cultivadas , Clonación Molecular , Conectina , ADN Complementario/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/metabolismo , Inmunoglobulinas/química , Datos de Secuencia Molecular , Miocardio/metabolismo , Fosfatos/química , Fosfatidilinositoles/química , Fosfolípidos/química , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Sarcómeros/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.