RESUMO
Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.
Assuntos
Acer/metabolismo , Inibidores da Angiogênese/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Hep G2 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica/patologia , Neovascularização Patológica/prevenção & controle , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Low-level laser therapy/treatment (LLLT) using a minimally invasive laser needle system (MILNS) might enhance bone formation and suppress bone resorption. In this study, the use of 405 nm LLLT led to decreases in bone volume and bone mineral density (BMD) of tibial trabecular bone in wild-type (WT) and Per2 knockout (KO) mice. Bone volume and bone mineral density of tibial trabecular bone was decreased by 405 nm LLLT in Per2 KO compared to WT mice at two and four weeks. To determine the reduction in tibial bone, mRNA expressions of alkaline phosphatase (ALP) and Per2 were investigated at four weeks after 405 nm laser stimulation using MILNS. ALP gene expression was significantly reduced in the LLLT-stimulated right tibial bone of WT and Per2 KO mice compared to the non-irradiated left tibia (p < 0.001). Per2 mRNA expression in WT mice was significantly reduced in the LLLT-stimulated right tibial bone compared to the non-irradiated left tibia (p < 0.001). To identify the decrease in tibial bone mediated by the Per2 gene, levels of runt-related transcription factor 2 (Runx2) and ALP mRNAs were determined in non-irradiated WT and Per2 KO mice. These results demonstrated significant downregulation of Runx2 and ALP mRNA levels in Per2 KO mice (p < 0.001). Therefore, the reduction in tibial trabecular bone resulting from 405 nm LLLT using MILNS might be associated with Per2 gene expression.
Assuntos
Densidade Óssea , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Terapia com Luz de Baixa Intensidade , Proteínas Circadianas Period/genética , Tíbia/metabolismo , Tíbia/patologia , Animais , Osso e Ossos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Proteínas Circadianas Period/metabolismo , Tíbia/efeitos da radiação , Microtomografia por Raio-XRESUMO
While dimensional change under thermal loading dictates various device failure mechanisms in soft materials, the interplay between microstructures and thermal expansion remains underexplored. Here, we develop a novel method to directly probe the thermal expansion for nanoscale polymer films using an atomic force microscope as well as confining active thermal volume. In a model system, spin-coated poly(methyl methacrylate), we find that the in-plane thermal expansion is enhanced by 20-fold compared to that along the out-of-plane directions in confined dimensions. Our molecular dynamics simulations show that the collective motion of side groups along backbone chains uniquely drives the enhancement of thermal expansion anisotropy of polymers in the nanoscale limit. This work unveils the intimate role of the microstructure of polymer films on its thermal-mechanical interaction, paving a route to judiciously enhance the reliability in a broad range of thin-film devices.
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Most living organisms exhibit circadian rhythms in physiology and behavior. These oscillations are generated by an endogenous circadian clock and control many biological processes. Ceramide has attracted attention as a signal mediator in diverse cell processes including cell death and differentiation. The relationships between ceramide expression levels and the circadian clock have not previously been investigated. To determine if there are circadian variations in the content of ceramide, we measured ceramide concentrations in the livers of wild-type (WT) and mPer1/mPer2 double knockout (DKO) mice. The ceramide concentration in WT mice was dramatically increased at Zeitgeber Time 9 (ZT9; 9 h after lights-on time) and ZT21 but no rhythmicity in ceramide expression was seen in DKO mice. Because ceramide can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase), or by ceramide synthase (CerS)-mediated synthesis, we assayed the expression patterns of ceramide-related genes using real-time PCR. CerS2 expression levels showed a biphasic pattern of expression in WT mice but no rhythmicity in DKO mice. While the neutral SMase (nSMase) and acidic SMase (aSMase) mRNA in WT mice were expressed in a circadian manner, the correlation between the expression levels of these SMases with times of day was weak in DKO mice. Collectively, our findings suggest that both SMases and CerS2 mRNA expression are regulated by the presence of mPer1/mPer2 circadian clock genes in vivo, and imply that ceramide may play a vital role in circadian rhythms and physiology.
Assuntos
Ceramidas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Circadianas Period/deficiência , Animais , Ritmo Circadiano/genética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Circadianas Period/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismo , Fatores de TempoRESUMO
Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [(3)H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.
Assuntos
Ciclo Celular/efeitos dos fármacos , Ceramidas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Neointima/patologia , Animais , Aorta/citologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Neointima/induzido quimicamente , Neointima/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Among the key metabolites produced by probiotic lactic acid bacteria (LAB), the use of gamma-aminobutyric acid (GABA), which alleviates hypertension, depression, and sleepiness in humans, is gaining popularity. Thus, GABA-producing LAB are sought after. GABA-producing LAB were preliminarily screened in acidified-MRS broth and quantified via GABase assays. The one-factor-at-a-time strategy was applied to determine the optimal conditions for GABA production. GABA production in reconstituted skim milk medium (RSM) and antibiotic susceptibility testing were performed to evaluate the potential of the strain as a yogurt starter. L. plantarum Y7 produced 4,856.86 ± 82.47 µg/mL of GABA at optimal culture conditions. Co-cultivation of Y7 and commercial Lactobacillus bulgaricus affected the amount of GABA production (6.85 ± 0.20 µg/mL) in RSM. Y7 was susceptible to ampicillin, erythromycin, and tetracycline. Therefore, L. plantarum Y7 represents a promising strain for GABA production in the food industry.
RESUMO
The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.
Assuntos
Ritmo Circadiano/genética , Estresse Oxidativo/genética , Proteínas Circadianas Period/genética , Superóxido Dismutase/biossíntese , Animais , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fígado/enzimologia , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1 , terc-Butil Hidroperóxido/farmacologiaRESUMO
The suprachiasmatic nuclei in the mammalian brain function as the regulators of circadian rhythm and coordinate the peripheral oscillators. Losses of clock genes alter gene expression and behavior. Here, we investigated whether disruption of the circadian clock and glucocorticoid signals would influence the gene expression of major urinary protein (Mup) in mice. Both Mup2 mRNA and protein showed biphasic rhythms with similar phase relationships. However, the peak of the rhythm is shifted in mPeriod2 circadian clock mutant mice. We identified two E-boxes and one glucocorticoid response element (GRE) as regulatory elements for Mup2 transcription. While CLOCK binds to the E-boxes constantly, glucocorticoid receptor was capable of binding to the GRE in a timely manner. All together, our results indicate that Mup2 expression is regulated by both the circadian clock and glucocorticoid.
Assuntos
Relógios Circadianos/fisiologia , Regulação da Expressão Gênica , Glucocorticoides/fisiologia , Proteínas/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/metabolismo , Imunoprecipitação da Cromatina , Relógios Circadianos/genética , Glucocorticoides/farmacologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Células NIH 3T3 , Proteínas Circadianas Period/genética , Multimerização Proteica , Transcrição GênicaRESUMO
We investigated the effects of sauchinone, isolated from the root of Saururus chinensis, on muscle disorders and the underlying mechanism of oxidative stress-induced C(2)C(12) skeletal muscle myoblast damage. To assess the protective effects of sauchinone on oxidative stress-induced C(2)C(12) skeletal muscle myoblasts, we measured the viability of the cells, showing that sauchinone pre-treatment significantly reduced the decreased cell viability after H(2)O(2) treatment. We also investigated the mechanism of this protective effect of sauchinone. In Western blot analysis, the heat shock protein (HSP)-70 level increased significantly in the sauchinone-pretreated myoblasts. We used high performance liquid chromatography (HPLC) to examine the level of endogenous ceramide after pre-treatment with sauchinone followed by exposure to H(2)O(2). While hydrogen peroxide increased the ceramide content to approximately 166.60±38.93% of the control level, pre-treatment with sauchinone inhibited this increase, maintaining the ceramide content at the control level. We demonstrated that sauchinone regulates intracellular HSP70 expression as well as ceramide levels to protect against oxidative stress-induced C(2)C(12) muscle myoblast damage. We suggest the potential benefits of herbal medicines in the treatment of oxidative stress-related muscle disorders.
Assuntos
Antioxidantes/farmacologia , Benzopiranos/farmacologia , Ceramidas/metabolismo , Dioxóis/farmacologia , Mioblastos Esqueléticos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Saururaceae/química , Antioxidantes/uso terapêutico , Benzopiranos/uso terapêutico , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dioxóis/uso terapêutico , Regulação para Baixo , Proteínas de Choque Térmico HSP70/metabolismo , Peróxido de Hidrogênio , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/patologia , Fitoterapia , Extratos Vegetais/uso terapêutico , Raízes de PlantasRESUMO
Transmission electron microscopy (TEM) is arguably the most important tool for atomic-scale material characterization. A significant portion of the energy of transmitted electrons is transferred to the material under study through inelastic scattering, causing inadvertent damage via ionization, radiolysis, and heating. In particular, heat generation complicates TEM observations as the local temperature can affect material properties. Here, the heat generation due to electron irradiation is quantified using both top-down and bottom-up approaches: direct temperature measurements using nanowatt calorimeters as well as the quantification of energy loss due to inelastic scattering events using electron energy loss spectroscopy. Combining both techniques, a microscopic model is developed for beam-induced heating and to identify the primary electron-to-heat conversion mechanism to be associated with valence electrons. Building on these results, the model provides guidelines to estimate temperature rise for general materials with reasonable accuracy. This study extends the ability to quantify thermal impact on materials down to the atomic scale.
RESUMO
In this work, we evaluated the oxygen evolution performance of cobalt oxide (CoO x)-coated carbon fiber paper in electrochemical water splitting. For a uniform coating of CoO x layers along the carbon fiber paper, the atomic layer deposition (ALD) technique was applied. We achieved a uniform and conformal coating of atomic-layer-deposited CoO x (ALD-CoO x) on the carbon fiber paper. The overpotential for oxygen evolution measured for the optimized ALD-coated carbon fiber paper was as low as 343 mV at 10 mA cm-2, which is competitive with the activity of state-of-the-art CoO x prepared on electrodes with large surface areas. Oxygen evolution is not enhanced after a critical thickness, about 28 nm in our study, is reached. The optimal thickness of the ALD-CoO x film is dependent on two competing effects: the high oxidation state of cobalt ions in thicker CoO x helps the oxygen evolution, whereas the introduction of a thick oxide coating decelerates the rate of charge transfer at the surface.
RESUMO
In the Drosophila circadian clock, daily cycles in the RNA levels of dclock (dClk) are antiphase to those of period (per). We altered the timing/levels of dClk expression by generating transgenic flies whereby per circadian regulatory sequences were used to drive rhythmic transcription of dClk. The results indicate that posttranscriptional mechanisms make substantial contributions to the temporal changes in the abundance of the dCLK protein. Circadian regulation is largely unaffected in the transgenic per-dClk flies despite higher mean levels of dCLK. However, in per-dClk flies the duration of morning activity is lengthened in light-dark cycles and light pulses evoke longer lasting bouts of activity. Our findings suggest that, in addition to a role in generating circadian rhythms, dCLK modulates the direct effects of light on locomotion.
Assuntos
Proteínas de Drosophila/genética , Luz , Atividade Motora/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas CLOCK , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
The 1st circadian "clock" gene identified was the X-linked period (per) gene in Drosophila melanogaster. In the pioneering initial report, Konopka and Benzer (1971) characterized 3 alleles of per that shortened (per (S); approximately 19 h), lengthened (per (L); approximately 29 h), or abolished (per (0)) circadian behavioral rhythms. They also showed that transheterozygotes carrying the per (S) and per (L) mutations exhibit robust behavioral rhythms with nearly normal periods of approximately 23 h, highlighting the semidominant nature of many clock mutants. In this study, per (0) flies bearing a doubly mutated per transgene that carries both the per (S) and per (L) alleles (per (0); per (S/L)) were analyzed for behavioral and molecular rhythms. Unlike singly mutated versions, the per (0);per ( S/L) transgenic flies are arrhythmic in constant dark conditions and exhibit little, if any, entrainment to daily light-dark cycles. In a wildtype per (+) background, expression of per ( S/L) abolishes behavioral rhythms, indicating that it functions in a transdominant negative fashion. Biochemical analysis of head extracts revealed that only hyperphosphorylated isoforms of the PERS/L protein are detected throughout a daily cycle, and the levels remain constant. Intriguingly, little if any PERS/L is observed in key pacemaker neurons that control daily activity rhythms, consistent with the notion that hyperphosphorylated isoforms of PER are unstable. Nonetheless, PERS/L is detected in ectopic cells in the brain, in which it exhibits an unusual localization, mainly staining the periphery of the nucleus. These results suggest that posttranslational mechanisms play a key role in limiting the accumulation of PER to specific cells. On a broader scope, our results indicate that the semidominant effects of period-altering alleles observed in trans are not necessarily preserved in the cis-configuration and that novel phenotypes can emerge.
Assuntos
Encéfalo/fisiologia , Ritmo Circadiano/genética , Drosophila/fisiologia , Mutação , Proteínas Nucleares/genética , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Técnicas de Cultura de Células , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Homeostase , Proteínas Circadianas Period , Fosforilação , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Anabaena sensory rhodopsin is a seven transmembrane protein that uses all-trans/13-cis retinal as a chromophore. About 22 residues in the retinal-binding pocket of microbial rhodopsins are conserved and important to control the quality of absorbing light and the function of ion transport or sensory transduction. The absorption maximum is 550 nm in the presence of all-trans retinal at dark. Here, we mutated Pro206 to Glu or Asp, of which the residue is conserved as Asp among all other microbial rhodopsins, and the absorption maximum and pKa of the proton acceptor group were measured by absorption spectroscopy at various pHs. Anabaena rhodopsin was expressed best in Escherichia coli in the absence of extra leader sequence when exogenous all-trans retinal was added. The wild-type Anabaena rhodopsin showed small absorption maximum changes between pH 4 and 11. In addition, Pro206Asp showed 46 nm blue-shift at pH 7.0. Pro206Glu or Asp may change the contribution to the electron distribution of the retinal that is involved in the major role of color tuning for this pigment. The critical residue Ser86 (Asp 96 position in bacteriorhodopsin: proton donor) for the pumping activity was replaced with Asp, but it did not change the proton pumping activity of Anabaena rhodopsin.
Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Rodopsinas Sensoriais/química , Rodopsinas Sensoriais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Anabaena/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , DNA Bacteriano/genética , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Bombas de Próton/química , Bombas de Próton/genética , Bombas de Próton/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldeído/metabolismo , Rodopsinas Sensoriais/genética , Homologia de Sequência de Aminoácidos , EspectrofotometriaRESUMO
In this work, we have successfully fabricated lanthanum strontium cobalt ferrite (LSCF)-gadolinium-doped ceria (GDC) composite cathodes by inkjet printing and demonstrated their functioning in solid oxide fuel cells (SOFCs). The cathodes are printed using a low-cost HP inkjet printer, and the LSCF and GDC source inks are synthesized with fluidic properties optimum for inkjet printing. The composition and microstructure of the LSCF and GDC layers are successfully controlled by controlling the color level in the printed images and the number of printing cycles, respectively. Anode-support type SOFCs with optimized LSCF-GDC composite cathodes synthesized by our inkjet printing method have achieved a power output of over 570 mW cm-2 at 650 °C, which is comparable to the performance of a commercial SOFC stack. Electrochemical impedance analysis is carried out to establish a relationship between the cell performance and the compositional and structural characteristics of the printed LSCF-GDC composite cathodes.
RESUMO
In reducing the high operating temperatures (≥800 °C) of solid-oxide fuel cells, use of protonic ceramics as an alternative electrolyte material is attractive due to their high conductivity and low activation energy in a low-temperature regime (≤600 °C). Among many protonic ceramics, yttrium-doped barium zirconate has attracted attention due to its excellent chemical stability, which is the main issue in protonic-ceramic fuel cells. However, poor sinterability of yttrium-doped barium zirconate discourages its fabrication as a thin-film electrolyte and integration on porous anode supports, both of which are essential to achieve high performance. Here we fabricate a protonic-ceramic fuel cell using a thin-film-deposited yttrium-doped barium zirconate electrolyte with no impeding grain boundaries owing to the columnar structure tightly integrated with nanogranular cathode and nanoporous anode supports, which to the best of our knowledge exhibits a record high-power output of up to an order of magnitude higher than those of other reported barium zirconate-based fuel cells.
RESUMO
Much progress has been made in understanding the molecular underpinnings governing circadian ( approximately 24 h) rhythms. Despite the increased complexity in metazoans whereby inter-cellular networks form the basis for driving overt rhythms, such as wake-sleep cycles in animals, single isolated cells can exhibit all the formal properties of a circadian pacemaker. How do these cell-autonomous rhythm generators operate? Breakthrough studies in Drosophila melanogaster led to the realization that the molecular logic underlying circadian clocks are highly shared. Most notably, interconnected transcriptional-translational feedback loops produce coordinated rhythms in "clock" RNAs and proteins that are required for the daily progression of clocks, synchronization to local time and transducing temporal signals to downstream effector pathways. More recent findings indicate prominent roles for reversible phosphorylation of clock proteins in the core oscillatory mechanism. In this review we focus on findings in Drosophila to explore the multiple levels that reversible phosphorylation plays in clock function. Specific clock proteins in this system are subjected to different phosphorylation programs, which affect three key properties of a circadian oscillator, its period, amplitude and phase. The role of phosphorylation in clocks is of clear relevance to human health because mutations that affect the PERIOD (PER) phosphorylation program are associated with familial sleep disorders. In addition, the central role of phosphorylation in the assembly of a circadian oscillator was dramatically shown recently by the ability to reconstitute a circadian phosphorylation/dephosphorylation cycle in vitro, suggesting that the dynamics of clock protein phosphorylation are at the "heart" of circadian time-keeping.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Retroalimentação Fisiológica/fisiologia , Animais , Proteínas CLOCK , Caseína Quinase 1 épsilon/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Proteínas Nucleares/genética , Proteínas Circadianas Period , Fosforilação , Biossíntese de Proteínas/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Unloading of skeletal muscle causes atrophy and altered contractility. To identify major muscle proteins responding significantly to the altered loading and to elucidate how the contractile alterations reflect potential proteomic modifications, we examined protein expression in the rat soleus muscle during 3-week hindlimb suspension and 2-week reloading. Compared with unsuspended controls, experimental animals had a 0.5- to 0.6-fold decrease in tension during unloading and early reloading, comparable to 0.2- to 0.6-fold decreases in the protein levels of myosin light chain 1 (MLC1), alpha-actin, tropomyosin beta-chain, and troponins T1 and T2. The observed 1.4- to 1.6-fold increase in shortening velocity appears to reflect 1.2- to 9.0-fold increases in the protein levels of fast-type MLC2, glycolytic enzymes, and creatine kinase, and 0.2- to 0.3-fold decreases in slow-type troponins T1 and T2. The levels of three heat shock proteins (p20, alpha crystallin B chain, and HSP90) decreased during unloading but returned to control levels during reloading. These results imply that proteomic responses to unloading change overall myofibrillar integrity and metabolic regulation, resulting in altered contractility.
Assuntos
Elevação dos Membros Posteriores , Contração Muscular , Músculo Esquelético/metabolismo , Proteômica , Animais , Proteínas Sanguíneas/metabolismo , Proteínas de Choque Térmico/metabolismo , Músculo Esquelético/fisiologia , Atrofia Muscular , Ratos , Ratos Sprague-DawleyRESUMO
The molecular components that generate and maintain circadian rhythms of physiology and behavior in mammals are present both in the brain (suprachiasmatic nucleus; SCN) and in peripheral tissues. Examination of mice with targeted disruptions of either mPer1 or mPer2 has shown that these two genes have key roles in the SCN circadian clock. Here we show that loss of the clock gene mPer2 affects forced locomotor performance in mice without altering muscle contractility. A proteomic analysis revealed that the anterior tibialis muscles of the mPer2 knockout mice had higher levels of glycolytic enzymes such as triose phosphate isomerase and enolase than those of either the wild type or mPer1 knockout mice. In addition, the level of expression of HSP90 in the mPer2 mutant mice was also significantly higher than in wildtype mice. These results suggest that the reduced locomotor endurance of the mPer2 knockout mice reflects a greater dependence on anaerobic metabolism under stress conditions, and that the two canonical clock genes, mPer1 and mPer2, play distinct roles in the physiology of skeletal muscle.
Assuntos
Relógios Biológicos , Proteínas de Ciclo Celular/genética , Músculo Esquelético/fisiologia , Proteínas Nucleares/genética , Proteômica , Fatores de Transcrição/genética , Animais , Proteínas Sanguíneas/metabolismo , Ritmo Circadiano , Proteínas Contráteis/metabolismo , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Atividade Motora , Contração Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/química , Proteínas Circadianas PeriodRESUMO
In this study, we used a compositionally gradient anode functional layer (AFL) consisting of Ni-BaCe(0.5)Zr(0.35)Y(0.15)O(3-δ) (BCZY) with increasing BCZY contents toward the electrolyte-anode interface for high-performance protonic ceramic fuel cells. It is identified that conventional homogeneous AFLs fail to stably accommodate a thin film of BCZY electrolyte. In contrast, a dense 2 µm thick BCZY electrolyte was successfully deposited onto the proposed gradient AFL with improved adhesion. A fuel cell containing this thin electrolyte showed a promising maximum peak power density of 635 mW cm(-2) at 600 °C, with an open-circuit voltage of over 1 V. Impedance analysis confirmed that minimizing the electrolyte thickness is essential for achieving a high power output, suggesting that the anode structure is important in stably accommodating thin electrolytes.