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Parasitic lifestyle can often relax the constraint on the plastome, leading to gene pseudogenization and loss, and resulting in diverse genomic structures and rampant genome degradation. Although several plastomes of parasitic Cuscuta have been reported, the evolution of parasitism in the family Convolvulaceae which is linked to structural variations and reduction of plastome has not been well investigated. In this study, we assembled and collected 40 plastid genomes belonging to 23 species representing four subgenera of Cuscuta and ten species of autotrophic Convolvulaceae. Our findings revealed nine types of structural variations and six types of inverted repeat (IR) boundary variations in the plastome of Convolvulaceae spp. These structural variations were associated with the shift of parasitic lifestyle, and IR boundary shift, as well as the abundance of long repeats. Overall, the degradation of Cuscuta plastome proceeded gradually, with one clade exhibiting an accelerated degradation rate. We observed five stages of gene loss in Cuscuta, including NAD(P)H complex â PEP complex â Photosynthesis-related â Ribosomal protein subunits â ATP synthase complex. Based on our results, we speculated that the shift of parasitic lifestyle in early divergent time promoted relaxed selection on plastomes, leading to the accumulation of microvariations, which ultimately resulted in the plastome reduction. This study provides new evidence towards a better understanding of plastomic evolution, variation, and reduction in the genus Cuscuta.
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Convolvulaceae , Cuscuta , Genoma de Plastidios , Convolvulaceae/genética , Cuscuta/genética , Genes de Plantas , Fotosíntesis/genética , Filogenia , Evolución MolecularRESUMEN
Acute kidney injury (AKI) is a major clinical problem associated with high morbidity and mortality. Esculentoside A (EsA), a kind of saponin isolated from the root of the Chinese herb Phytolaca esculenta, has been reported to have anti-inflammatory effect. In this study, we aimed to investigate the protective effects of EsA on LPS-induced AKI in mice. The protective effects of EsA was evaluated by detecting kidney histological change, blood urea nitrogen (BUN) and creatinine levels, and inflammatory cytokines production. The results showed that EsA significantly attenuated LPS-induced kidney histological change, as well as BUN and creatinine levels. EsA also inhibited LPS-induced TNF-α, IL-1ß, and IL-6 production. LPS-induced NF-κB activation was significantly suppressed by treatment of EsA. In addition, EsA up-regulated the expression of PPAR-γ in a dose-dependent manner. In conclusion, EsA protected mice effectively from LPS-induced AKI by PPAR-γ, which subsequently inhibited LPS-induced inflammatory response.
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Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Lipopolisacáridos/efectos adversos , Ácido Oleanólico/análogos & derivados , PPAR gamma/metabolismo , Saponinas/antagonistas & inhibidores , Lesión Renal Aguda/patología , Animales , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Ácido Oleanólico/administración & dosificación , Ácido Oleanólico/antagonistas & inhibidores , Saponinas/administración & dosificación , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
UNLABELLED: The present study aimed to assess the prognostic value of early amplitude-integrated electroencephalogram (aEEG) in late preterm infants who were born at a gestational age between 34 0/7 and 36 6/7 weeks for the prediction of neurobehavioral development. Late preterm infants (n = 170) with normal, mild, and severe asphyxia underwent continuous recording of aEEG for 4-6 h starting 6-8 h after delivery. The recordings were analyzed for background pattern, sleep-wake cycle (SWC), and seizures. Survivors were assessed at 18 months by neurological examination and Bayley Scales of Infant Development II. The incidence of adverse neurological outcome in the asphyxia group was significantly higher than in the normal group. For late preterm infants in the asphyxia group, abnormal aEEG pattern had a predictive potential of neurological outcomes with sensitivity of 78.57% (specificity, 87.80%; positive predictive value [PPV], 68.75%; negative predictive value [NPV], 92.31%; power, 85.45%). Non-SWC and intermediate SWC significantly were increased (25.45 and 52.73%, respectively) in the asphyxia group vs. the normal group. SWC pattern had neurological prognosis value in the asphyxia group with sensitivity of 64.29% (specificity, 87.80%; PPV, 64.29%; NPV, 87.80%; power, 81.82%). CONCLUSION: Early aEEG patterns are important determinants of long-term prognosis of neurodevelopmental outcome in asphyxiated late preterm infants.
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Asfixia Neonatal/fisiopatología , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Electroencefalografía , Recien Nacido Prematuro/crecimiento & desarrollo , Femenino , Edad Gestacional , Humanos , Lactante , Recién Nacido , Masculino , Examen Neurológico/métodos , Valor Predictivo de las Pruebas , Embarazo , Pronóstico , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The suspended particle size distribution provides crucial information for the study of water environment structure and function. Based on the in-situ data from wet and dry season in 2008-2011, the paper studied suspended particle size distribution of Poyang Lake and its optical features. The suspended particle size distribution showed seasonal variation: the particle size of southern lake was larger than that of northern lake in dry season but showed little variation in wet season. The suspended particle size distribution exerted influence on particulate absorption coefficient, attenuation coefficient and scattering coefficient. The particulate absorption coefficient of northern lake was higher than that of southern lake. The negative correlation between specific absorption coefficient of total suspended particles and median particle size indicated that there was "package effect" of mineral particles in turbid Poyang Lake. The spatial and temporal distribution of particulate attenuation coefficient and scattering coefficient are similar: there were obvious regional differences in dry season but few in wet season. There were good correlations among the remote sensing reflectance, spectra slope of particle size distribution and spectra slope of particulate scattering coefficient. These correlations would provide the foundation for remote sensing retrieval of particle size and quantitative analysis of influence of suspended particle size on the optical properties. The relationship between particle size distribution, particulate back-scattering coefficient and bulk refractive index can provide information of particle composition in Poyang Lake.
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A model for partitioning the particulate scattering coefficient into the contributions of suspended mineral particle and organic particle was proposed based on the measured data. The independent variables, i. e. the concentrations of mineral particles and organic particles in this study, were used to determine the mass-specific scattering cross section with the concurrent total suspended particulate scattering coefficients collected during the field trip in Poyang Lake 2009. Results show that the scattering spectra of inorganic particles and organic particles can be successful derived by the proposed model, and the reconstructed total particulate scattering coefficients are in better agreement with the measured values by the ordinary least square linear regression. For the whole South Poyang Lake, mean absolute percentage errors between the measured scattering coefficients and reconstructed value were less than 25% over the main remote sensing effective wavebands such as 440, 532, 555 and 676 nm. A remarkable lower predicted error, which can be controlled within 15%, were found at all stations with higher concentration of total suspended matters, while the spectral partitioning is less efficient at stations with total suspended particle concentration less than 15 mg x L(-1). Particulate scattering spectrum retrieved by RMA shows that illite and montmorillonite are the major constituents of inorganic matters which dominate the light scattering properties of Poyang Lake. It is possible that scattering spectrum partitioned by the model could infer the major effective components in waters, and could be used to predict particulate scattering properties for highly turbid waters.
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A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.
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Regulación Neoplásica de la Expresión Génica , Neoplasias Intestinales/genética , Mutación , Cadenas Pesadas de Miosina/genética , Miosina Tipo II/genética , Miosinas del Músculo Liso/genética , Poliposis Adenomatosa del Colon/genética , Secuencia de Aminoácidos , Estudios de Casos y Controles , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN , Humanos , Datos de Secuencia Molecular , Síndrome de Peutz-Jeghers/genética , Miosinas del Músculo Liso/fisiologíaRESUMEN
OBJECTIVE: This study examined the effect of recombinant human erythropoietin (r-HuEPO) on the serum levels of neuron-specific enolase (NSE), S-100ß protein and myelin basic protein (MBP) in young rats 24 hrs after lithium-pilocarpine-induced status epilepticus (SE) in order to study the potential role of r-HuEPO in epileptic brain damage. METHODS: Forty 19-21-day-old male Sprague-Dawley (SD) rats were randomly divided into four groups (n=10): normal control group, SE, r-HuEPO pretreated-SE and r-HuEPO. SE was induced by lithium-pilocarpine. R-HuEPO (500 IU/kg) was intraperitoneally injected in the r-HuEPO pretreated-SE and r-HuEPO groups 4 hrs before SE. Serum levels of NSE, S-100ß and MBP were determined 24 hrs after the SE event. RESULTS: Serum levels of NSE, S-100ß and MBP in the SE group increased significantly compared with those in the normal control and the r-HuEPO groups (Pï¼0.05). The r-HuEPO pretreated-SE group showed significantly decreased serum levels of NSE, S-100ß and MBP compared with the SE group (Pï¼0.05). CONCLUSIONS: r-HuEPO may reduce the expression of NSE, S-100ß and MBP and thus might provide an early protective effect against epileptic brain injury.
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Eritropoyetina/uso terapéutico , Proteína Básica de Mielina/sangre , Factores de Crecimiento Nervioso/sangre , Fosfopiruvato Hidratasa/sangre , Proteínas S100/sangre , Estado Epiléptico/tratamiento farmacológico , Animales , Eritropoyetina/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Subunidad beta de la Proteína de Unión al Calcio S100 , Estado Epiléptico/sangreRESUMEN
BACKGROUND: Chronic insomnia is a major public health problem, but there are limited effective therapies. Jiawei Suanzaoren Decoction (JW-SZRD) has been used as an alternative option for treating insomnia. This study aimed to investigate the long-term efficacy and safety of JW-SZRD in combination with lorazepam for chronic insomnia. METHODS: A total of 207 participants were analyzed in this study. The treatment group (TG) received JW-SZRD and lorazepam orally, and the control group (CG) received lorazepam alone. The Insomnia Severity Index (ISI), the Self-Rating Depression Scale (SDS), the Self-Rating Anxiety Scale (SAS), and the Somatic Self-rating Scale (SSS) were evaluated at baseline, weeks 4, 8, and 12. The MOS 36-item Short Form Health Survey (SF-36) was assessed at baseline and week 12. Adverse effects (AEs) were evaluated by the Treatment Emergent Symptom Scale (TESS). RESULTS: Both TG and CG showed obvious improvements in the sleep onset latency (SOL) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (d = 1.28). The ISI reduction rate in TG was higher than that in CG at weeks 4, 8, and 12 (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (P=0.001 and 0.005) and total sleep time (TST) (. CONCLUSION: The combination of JW-SZRD with lorazepam can significantly improve sleep quality with fewer AEs. It is an effective treatment and superior to lorazepam alone for chronic insomnia.
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AIM: To study the effect of modified starfish sterol [C03, succinic acid (5-epiandroene-17-one-3beta-ol) diester] on experimental arrhythmias. METHODS: Arrhythmias were induced by drugs (Aco, Oua, BaCl2 and adrenalin) i.v., ligating the left anterior descending coronary artery and electricity. RESULTS: C03 71.4 mg x kg(-1) (ig) was shown to increase the dose of Oua inducing VP, VT, VF and CA in guinea pigs (P < 0.01); C03 (26.8, 80.4 mg x kg(-1)) was found to increase the dose of Aco inducing VF and CA in rats (P < 0.01); C03 (8.9, 26.8, 80.4 mg x kg(-1)) increase the dose of barium chloride and delay the onset time of ventricular arrhythmias (P < 0.01); C03 (14.1, 42.3 mg x kg(-10) shorten time of recovering induced by adrenalin in rabbits (P < 0.01); C03 (80.4 mg x kg(-1)) was shown to reduce the number of ventricular arrhythmias induced by coronary artery ligation in rats (P < 0.05), C03 increase VFT induced by electricity in rabbits, VFT of C03 14.1 mg x kg(-1) increased from (5.1 +/- 2.5) V to (11.0 +/- 2.7) V (P < 0.01), 42.3 mg x kg(-1) increased from (6.1 +/- 1.7) V to (15 +/- 5) V (P < 0.01). CONCLUSION: Starfish sterol has anti-arrhythmic effect.
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Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/prevención & control , Materia Medica/uso terapéutico , Estrellas de Mar , Esteroles/uso terapéutico , Aconitina , Animales , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/fisiopatología , Compuestos de Bario , Gatos , Cloruros , Epinefrina , Cobayas , Materia Medica/aislamiento & purificación , Ratones , Ouabaína , Conejos , Ratas , Estrellas de Mar/química , Esteroles/aislamiento & purificación , Fibrilación Ventricular/fisiopatologíaRESUMEN
OBJECTIVE: To screen and identify differentially expressed proteins of mesenchymal stem cells (MSC) during endothelial differentiation. METHODS: MSCs were induced to endothelial differentiation with vascular endothelial growth factor (VEGF) and epithelial growth factor (EGF) mixture. The whole cell proteins were extracted and isolated by two-dimensional gel electrophoresis. After gel was analyzed by Imagemaster 5.0 software, differentially expressed proteins were partially selected and identified by MALDI-TOF-MS. RESULTS: The differentiated MSC highly expressed endothelial cells related markers, CD31, CD34 and FVIIIAg were 56.8%, 38.8% and 14.5% respectively by flow cytometer. Compared with the primary cultured MSC, the differentiated cells differentially expressed 91 proteins. Among the 19 identified proteins, 11 up-regulated and 8 down-regulated, which include cytoskeletal proteins, such as myosin, filamin, vimentin and vinculin; cell metabolism enzymes, such as ORP-150, ERO1-α, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, protein disulfide-isomerase A3, FAS and enolase 3; nuclear factors, such as TAR DNA binding protein, guanine nucleotide binding protein and hypoxia up-regulated protein 1; VEGF receptors, such as KDR and so on. CONCLUSIONS: Cytoskeletal proteins, metabolism enzymes and KDR were all involved in endothelial differentiation of MSC. These proteins may be the regulatory targets for endothelial differentiation of MSC.
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Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Células de la Médula Ósea/citología , Células Cultivadas , Células Endoteliales/citología , Masculino , Células Madre Mesenquimatosas/citología , Proteoma/análisis , Proteómica , Ratas , Ratas WistarRESUMEN
Myosin VI moves toward the pointed (minus) end of actin filaments, the reverse direction of other myosin classes. The myosin VI structure demonstrates that a unique insert at the end of the motor repositions its lever arm and is at least in part responsible for the reversal of directionality. However, it has been proposed that there must be additional modifications within the motor that contribute to its large step size and to the reversal of directionality. To ascertain the inherent directionality of the motor core, we attached the myosin V lever arm to myosin VI, with and without the unique insert. If the insert was maintained, the motor moved toward the minus end of actin filaments, but if removed, movement was redirected toward the plus end. Single-molecule studies revealed that further adaptations within the motor increase the magnitude and variability of the plus-end directed converter movements, and unexpectedly provide the source of the highly variable myosin VI step size. Thus, the unique insert is necessary and sufficient to reverse an inherently plus-end directed myosin.
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Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/metabolismo , Animales , Dimerización , Modelos Moleculares , Proteínas Motoras Moleculares/genética , Cadenas Pesadas de Miosina/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , PorcinosRESUMEN
Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. Herein, we demonstrate that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered, which move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, we present data consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104.
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Actinas/metabolismo , Cadenas Pesadas de Miosina , Estructura Cuaternaria de Proteína , Actinas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Dimerización , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/ultraestructura , PorcinosRESUMEN
Members of the myosin II class of molecular motors have been referred to as "conventional," a term used to describe their ability to form thick filaments, their low duty ratio, the ability of individual motor-containing "heads" to operate independently of each other, and their rate-limiting phosphate release. These features ensure that those motors that have completed their power stroke dissociate rapidly enough to prevent them from interfering with those motors that are beginning theirs. However, in this study, we demonstrate that myosin IIB, a cytoplasmic myosin II particularly enriched in the central nervous system and cardiac tissue, has a number of features that it shares instead with "unconventional" myosin isoforms, including myosins V and VI. These include a high duty ratio, rate-limiting ADP release, and high ADP affinity. These features imply that myosin IIB serves a set of physiologic needs different from those served by its more conventional myosin II counterparts, and this work provides a plausible basis for explaining the physiologic role of this unconventionally conventional myosin.
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Miosina Tipo IIB no Muscular/química , Miosina Tipo IIB no Muscular/fisiología , Actinas/química , Actinas/farmacología , Adenosina Difosfato/metabolismo , Animales , Línea Celular , Pollos , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Insectos , Cinética , Modelos Químicos , Fosfatos/química , Unión Proteica , Isoformas de Proteínas , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tends to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.
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Actinas/química , Leucina Zippers , Subfragmentos de Miosina/química , Miosina Tipo II/química , Adenosina Difosfato/química , Animales , Dimerización , Vectores Genéticos , Leucina Zippers/genética , Modelos Biológicos , Modelos Químicos , Músculo Esquelético/química , Músculo Liso/química , Subfragmentos de Miosina/genética , Miosina Tipo II/genética , Unión Proteica/genética , Conejos , Espectrometría de Fluorescencia , SpodopteraRESUMEN
Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of approximately 30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was approximately 60 nm, strongly supporting a hand-over-hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position, whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin.
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Actinas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Secuencia de Aminoácidos , Fluorescencia , Cadenas Pesadas de Miosina/química , Unión ProteicaRESUMEN
This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.
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Calcio/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Actinas/farmacología , Animales , Sitios de Unión , Calcio/farmacología , Cinética , Proteínas Motoras Moleculares , Movimiento (Física) , Cadenas Pesadas de Miosina/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Estructura Terciaria de Proteína , PorcinosRESUMEN
Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 A change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23 degrees swing of the light-chain domain.
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Actinas/química , Actinas/metabolismo , Miosinas/química , Miosinas/metabolismo , Sustitución de Aminoácidos , Animales , Pollos , Cisteína , Transferencia Resonante de Energía de Fluorescencia , Molleja de las Aves/metabolismo , Cinética , Músculo Liso/metabolismo , Mutagénesis Sitio-Dirigida , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Conformación ProteicaRESUMEN
Myosin VI contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single "IQ motif". Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, we demonstrate that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding, as truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. We suggest that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI "converter" region and repositions the myosin VI "lever arm" to allow reverse direction (minus-end) motility on actin.