959 resultados para Myosin regulatory light chain
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The 66 kilo-Dalton (k-Da) protein split off from the cross linked myosin heavy chain (CMHC) formed due to the setting of Alaska pollack surimi, frozen-storage of Pacific cod flesh, and vinegar-curing of Pacific mackerel mince was identified as a light meromyosin (LMM). Puncture and stress-relaxation tests showed that the actomyosin subunits (AMS) of Alaska pollack surimi, upon setting at 30°C, transformed into gel, although the elasticity of this gel was very low when compared to the gels from surimi or actomyosin (AM). Electrophoretic studies showed that the band due to LMM in the gel from AMS gradually disappeared with the progress of setting but higher molecular weight polymer did not form. The intensity of the bands due to other myosin sub-fragments decreased a little. The findings suggest that at setting temperature, LMM of MHC molecule leads to an unfolding resulting in an intramolecular aggregation through non-covalent interactions, and thus plays a significant role in the crosslinking of MHC.
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Two orange phosphorescent iridium complex monomers, 9-hexyl-9-(iridium (III)bis(2-(4'-fluorophenyl)-4-phenylquinoline-N, C-2')(tetradecanedionate-11,13))-2,7-dibromofluorene (Br-PIr) and 9-hexyl-9-(iridium(III)bis(2-(4'-fluorophenyl)-4-methylquinoline-N, C-2')(tetradecanedionate-11,13))-2,7-dibromofluorene (Br-MIr), were successfully synthesized. The Suzuki polycondensation of 2,7-bis(trimethylene boronate)-9,9-dioctylfluorene with 2,7-dibromo-9,9-dioetylfluorene and Br-Plr or Br-MIr afforded two series of copolymers, PIrPFs and MIrPFs, in good yields, in which the concentrations of the phosphorescent moieties were kept small (0.5-3 mol % feed ratio) to realize incomplete energy transfer. The photoluminescence (PL) of the copolymers showed blue- and orange-emission peaks. A white-light-emitting diode with a configuration of indium tin oxide/poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)/PIr05PF (0.5 mol % feed ratio of Br-PIr)/Ca/Al exhibited a luminous efficiency of 4.49 cd/A and a power efficiency of 2.35 lm/W at 6.0 V with Commission Internationale de L'Eclairage (CIE) coordinates of (0.46, 0.33). The CIE coordinates were improved to (0.34, 0.33) when copolymer MIr10PF (1.0 mol % feed ratio of Br-MIr) was employed as the white-emissive layer. The strong orange emission in the electroluminescence spectra in comparison with PL for these kinds of polymers was attributed to the additional contribution of charge trapping in the phosphorescent dopants.
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A series of blue light-emitting poly(aryl ether)s (PAEs) containing ter- or pentafluorenes in the main chain have been synthesized via nucleophilic substitution polycondensation reaction. The energy levels of the polymers were tuned by introducing hole-transporting triaryamine groups in the side chains and/or incorporating electron-transporting oxadiazole segments in the main chain. The optical properties of the polymers are dominantly determined by the well-defined oligofluorene segments, and therefore all polymers show high photoluminescence quantum yield. Differential scanning calorimeter (DSC) characterizations indicate that they are vitrified polymers with high glass transition temperature (up to 156 degrees C). The polymers comprising pentafluorenes exhibit electroluminescent properties equal to or better than fully conjugated fluorene homopolymers. With the device structure of ITO/PEDOT:PSS/polymer/Ca/Al, an external quantum efficiency of 1.4% along with Commission Internationale de L'Eclairage (CIE) coordinates of (0.17, 0.09) has
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A set of AM-AA copolymer samples with the same comonomer content and different average molecular weight have been characterized by C-13 NMB and light scattering methods in this paper. The chemical composition (comonomer AA, mole content 16.9 +/- 1.1%) of these samples is uniform. the sequence of AA in the macromolecular chain is of alone and random distribution and the light scattering theory from polyelectrolyte in added-salt solutions is suitable for the AM-AA copolymers-0.12 mol/L NaCl water systems. The actual values of M(w), the second Virial coefficient A(2) and the mean square radius of gyration (R(2)), for the studied samples have been obtained. The relationships between the molecular parameters are as follows: A(2)=0.0619 ($) over bar M(w)(-0.24), < R(2) >(1/2)(t)= 0.0210 ($) over bar M(w)(0.54).
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.
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The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.
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The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.
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Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.
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Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ∼10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN–nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.
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The expression of desacetoxyvindoline 4-hydroxylase (D4H), which catalyzes the second to the last reaction in vindoline biosynthesis in Catharanthus roseus, appears to be under complex, multilevel developmental and light regulation. Developmental studies with etiolated and light-treated seedlings suggested that although light had variable effects on the levels of d4h transcripts, those of D4H protein and enzyme activity could be increased, depending on seedling development, up to 9- and 8-fold, respectively, compared with etiolated seedlings. However, light treatment of etiolated seedlings could stop and reverse the decline of d4h transcripts at later stages of seedling development. Repeated exposure of seedlings to light was also required to maintain the full spectrum of enzyme activity observed during seedling development. Further studies showed that a photoreversible phytochrome appeared to be involved in the activation of D4H, since red-light treatment of etiolated seedlings increased the detectable levels of d4h transcripts, D4H protein, and D4H enzyme activity, whereas far-red-light treatment completely reversed this process. Additional studies also confirmed that different major isoforms of D4H protein exist in etiolated (isoelectric point, 4.7) and light-grown (isoelectric point, 4.6) seedlings, suggesting that a component of the light-mediated activation of D4H may involve an undetermined posttranslational modification. The biological reasons for this complex control of vindoline biosynthesis may be related to the need to produce structures that could sequester away from cellular activities the cytotoxic vinblastine and vincristine dimers that are derived partially from vindoline.
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Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.