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1.
J Neurosci Res ; 98(3): 422-436, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-30957296

RESUMO

Although molecular tools for controlling neuronal activity by light have vastly expanded, there are still unmet needs which require development and refinement. For example, light delivery into the brain is still a major practical challenge that hinders potential translation of optogenetics in human patients. In addition, it would be advantageous to manipulate neuronal activity acutely and precisely as well as chronically and non-invasively, using the same genetic construct in animal models. We have previously addressed these challenges by employing bioluminescence and have created a new line of opto-chemogenetic probes termed luminopsins by fusing light-sensing opsins with light-emitting luciferases. In this report, we incorporated Chlamydomonas channelrhodopsin 2 with step-function mutations as the opsin moiety in the new luminopsin fusion protein termed step-function luminopsin (SFLMO). Bioluminescence-induced photocurrent lasted longer than the bioluminescence signal due to very slow deactivation of the mutated channel. In addition, bioluminescence was able to activate most of the channels on the cell surface due to the extremely high light sensitivity of the channel. This efficient channel activation was partly mediated by radiationless bioluminescence resonance energy transfer due to the proximity of luciferase and opsin. To test the utility of SFLMOs in vivo, we transduced the substantia nigra unilaterally via a viral vector in male rats. Injection of the luciferase substrate as well as conventional photostimulation via fiber optics elicited circling behaviors. Thus, SFLMOs expand the current approaches for manipulation of neuronal activity in the brain and add more versatility and practicality to optogenetics in freely behaving animals.


Assuntos
Channelrhodopsins , Luciferases , Neurônios/fisiologia , Optogenética/métodos , Animais , Comportamento Animal/fisiologia , Channelrhodopsins/genética , Channelrhodopsins/fisiologia , Feminino , Células HEK293 , Humanos , Luciferases/genética , Luciferases/fisiologia , Proteínas Luminescentes , Masculino , Potenciais da Membrana , Cultura Primária de Células , Ratos Sprague-Dawley , Substância Negra/fisiologia
2.
J Neurosci Res ; 98(3): 410-421, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-28862809

RESUMO

Previous work has demonstrated that fusion of a luciferase to an opsin, to create a luminescent opsin or luminopsin, provides a genetically encoded means of manipulating neuronal activity via both chemogenetic and optogenetic approaches. Here we have expanded and refined the versatility of luminopsin tools by fusing an alternative luciferase variant with high light emission, Gaussia luciferase mutant GLucM23, to depolarizing and hyperpolarizing channelrhodopsins with increased light sensitivity. The combination of GLucM23 with Volvox channelrhodopsin-1 produced LMO4, while combining GLucM23 with the anion channelrhodopsin iChloC yielded iLMO4. We found efficient activation of these channelrhodopsins in the presence of the luciferase substrate, as indicated by responses measured in both single neurons and in neuronal populations of mice and rats, as well as by changes in male rat behavior during amphetamine-induced rotations. We conclude that these new luminopsins will be useful for bimodal opto- and chemogenetic analyses of brain function.


Assuntos
Channelrhodopsins , Luciferases , Neurônios/fisiologia , Optogenética/métodos , Potenciais de Ação , Adenoviridae/fisiologia , Animais , Channelrhodopsins/genética , Channelrhodopsins/fisiologia , Feminino , Vetores Genéticos , Células HEK293 , Hipocampo/fisiologia , Humanos , Luciferases/genética , Luciferases/fisiologia , Masculino , Camundongos , Cultura Primária de Células , Ratos Sprague-Dawley , Volvox/genética
3.
J Neurosci Res ; 98(3): 437-447, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-30152529

RESUMO

In Bioluminescent Optogenetics (BL-OG) a biological, rather than a physical, light source is used to activate light-sensing opsins, such as channelrhodopsins or pumps. This is commonly achieved by utilizing a luminopsin (LMO), a fusion protein of a light-emitting luciferase tethered to a light-sensing opsin. Light of the wavelength matching the activation peak of the opsin is emitted by the luciferase upon application of its small molecule luciferin, resulting in activation of the fused opsin and subsequent effects on membrane potential. Using optimized protocols for culturing, transforming, and testing primary neurons in multi electrode arrays, we systematically defined parameters under which changes in neuronal activity are specific to bioluminescent activation of opsins, rather than due to off-target effects of either the luciferin or its solvent on neurons directly, or on opsins directly. We further tested if there is a direct effect of bioluminescence on neurons. Critical for assuring specific BL-OG effects are testing the concentration and formulation of the luciferin against proper controls, including testing effects of vehicle on LMO expressing and of luciferin on nonLMO expressing targets.


Assuntos
Luciferases , Medições Luminescentes , Neurônios/fisiologia , Opsinas , Optogenética/instrumentação , Optogenética/métodos , Animais , Eletrodos Implantados , Feminino , Luciferases/genética , Luciferases/fisiologia , Proteínas Luminescentes , Masculino , Potenciais da Membrana , Opsinas/genética , Opsinas/fisiologia , Cultura Primária de Células , Ratos Sprague-Dawley
5.
J Ethnopharmacol ; 237: 47-54, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30898554

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The quality control of Traditional Chinese medicine (TCM) is a scientific problem and an industrial issue, which hampers the development of evidence based TCM. The concept of quality markers (Q-markers) is proposed and has been applied to the quality evaluation of TCM based on its clinical efficacy. However, more specific methods are needed to put this idea into practice. The standard decoction is a representative of decoction used in clinical practice and it can be used for the discovery of Q-markers related to the clinical efficacy of TCM. AIM OF THE STUDY: In this study, a systemic strategy was established to discover Q-markers related to the clinical efficacy of TCM Ephedrae Herba (EH), dried stem of Ephedra sinica Stapf. The different processed materials of EH have different clinical applications, though originating from the same medicinal herb. MATERIALS AND METHODS: The standard decoction of each of the processed materials was prepared and a 1HNMR metabolomics approach and total polysaccharide analysis were used to identify potential Q-markers related to the different clinical applications of EH. Correlation analysis was made of the measured biological activity and the holistic chemical profile. RESULTS: The results showed that total polysaccharides and alkaloids were Q-markers for EH preparations. CONCLUSION: This study demonstrates that the standard decoction is a reasonable research objective to explore chemical markers that correlate with the clinical efficacy of TCM.


Assuntos
Ephedra , Extratos Vegetais/farmacologia , Caules de Planta , Controle de Qualidade , Células HEK293 , Humanos , Luciferases/fisiologia , Metabolômica , Regiões Promotoras Genéticas , Receptor 2 Toll-Like/fisiologia , Fator de Transcrição AP-1/fisiologia , Resultado do Tratamento
6.
Mol Biol (Mosk) ; 52(6): 935-947, 2018.
Artigo em Russo | MEDLINE | ID: mdl-30633237

RESUMO

The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH2 and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.


Assuntos
Aliivibrio/fisiologia , Luciferases/fisiologia , Photobacterium/fisiologia , Vibrio/fisiologia , Proteínas de Bactérias/fisiologia , DNA Bacteriano , Genes Bacterianos , Luminescência , Óperon
7.
Curr Opin Clin Nutr Metab Care ; 20(3): 175-180, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28376508

RESUMO

PURPOSE OF REVIEW: The article highlights the recent development of an ATF4 (activating transcription factor) inducible luciferase (LUC) mouse model to monitor the integrated stress response pathway (ISR) in vivo. RECENT FINDING: The ISR pathway plays a key role in cellular adaptation to stress and is dysregulated in numerous diseases. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 α, which leads to the recruitment of the transcription factor ATF4 to specific CCAAT/enhancer-binding protein-ATF response elements (CAREs) located in the promoters of target genes. To monitor the modulation of this pathway in the whole animal and at tissue and cellular levels, we generated a CARE-driven LUC mouse model. We validated the relevance of this model to study stress-related pathologies and recently observed the correlation between the ISR pathway induction in muscle and the occurrence of stress-induced skeletal muscle atrophy. SUMMARY: The CARE-LUC mouse model represents an innovative tool for investigating the role of the ISR pathway in physiology and disease and opens new avenues for the development of drugs that could modify this important pathway in stress-related human diseases.


Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Luciferases/fisiologia , Modelos Animais , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Animais , Camundongos , Músculo Esquelético/metabolismo , Atrofia Muscular/fisiopatologia , Fosforilação/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases
8.
J Neurosci ; 33(7): 2773-83, 2013 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-23407937

RESUMO

Neuroblastoma is an embryonic tumor derived from cells of the neural crest. Taking advantage of a newly developed neural crest lineage tracer and based on the hypothesis that the molecular mechanisms that mediate neural crest delamination are also likely to be involved in the spread of neuroblastoma, we were able to identify genes that are active both in neural crest development and neuroblastoma tumor formation. A subsequent search of the neuroblastoma gene server for human orthologues of genes differentially expressed in the chick embryo neural crest screen retrieved the LIM domain only protein 4 (LMO4), which was expressed in both cell types analyzed. Functional experiments in these two model systems revealed that LMO4 activity is required for neuroblastoma cell invasion and neural crest delamination. Moreover, we identified LMO4 as an essential cofactor in Snail2-mediated cadherin repression and in the epithelial-to-mesenchymal transition of both neural crest and neuroblastoma cells. Together, our results suggest that the association of high levels of LMO4 with aggressive neuroblastomas is dependent on LMO4 regulation of cadherin expression and hence, tumor invasiveness.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Neoplasias Encefálicas/patologia , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/fisiologia , Crista Neural/patologia , Neuroblastoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Western Blotting , Caderinas/biossíntese , Caderinas/fisiologia , Linhagem Celular Tumoral , Embrião de Galinha , DNA/genética , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Vetores Genéticos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Lentivirus/genética , Luciferases/fisiologia , Análise em Microsséries , Invasividade Neoplásica/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição da Família Snail , Timidina/metabolismo
9.
Am J Physiol Endocrinol Metab ; 304(3): E254-66, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23193055

RESUMO

To investigate in vivo transcription of the facilitative glucose transporter isoform-GLUT3 gene, we created GLUT3-firefly luciferase transgenic mouse lines that demonstrate tissue-specific [adult: brain > testis ≥ skeletal muscle > placenta; postnatal (PN): skeletal muscle > brain = skin], temporal, and spatial distribution of the reporter gene/enzyme activity that is unique from endogenous GLUT3 mRNA/protein. In this mouse model, luciferase expression/activity serving as a readout of in vivo transcription peaked at 12 days gestation along with proliferating cell nuclear antigen (cell replication) in placenta and embryonic brain preceding peak GLUT3 protein expression at 18-19 days gestation. In contrast, a postnatal increase in brain luciferase mRNA peaked with endogenous GLUT3 mRNA, but after that of NeuroD6 protein (neurogenesis) at PN7. Luciferase activity paralleled GLUT3 protein expression with Na(+)-K(+)-ATPase (membrane expansion) and synaptophysin (synaptogenesis) proteins, peaking at PN14 and lasting until 60 days in the adult. Thus GLUT3 transcription in placenta and embryonic brain coincided with cell proliferation and in postnatal brain with synaptogenesis. Longitudinal noninvasive bioluminescence (BLI) monitoring of in vivo brain GLUT3 transcription reflected cross-sectional ex vivo brain luciferase activity only between PN7 and PN21. Hypoxia/reoxygenation at PN7 revealed transcriptional increase in brain GLUT3 expression reflected by in vivo BLI and ex vivo luciferase activity. These observations collectively support a temporal contribution by transcription toward ensuring adequate tissue-specific, developmental (placenta and embryonic brain), and postnatal hypoxic brain GLUT3 expression.


Assuntos
Encéfalo/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Placenta/metabolismo , Ativação Transcricional/fisiologia , Transgenes/fisiologia , Animais , Feminino , Genes Reporter/fisiologia , Transportador de Glucose Tipo 3/genética , Luciferases/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Distribuição Tecidual
10.
J Neurosci ; 32(46): 16193-202, 2012 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-23152603

RESUMO

Aging produces a decline in the amplitude and precision of 24 h behavioral, endocrine, and metabolic rhythms, which are regulated in mammals by a central circadian pacemaker within the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. Disruption of the circadian system, as experienced during transmeridian travel, can lead to adverse health consequences, particularly in the elderly. To test the hypothesis that age-related changes in the response to simulated jet lag will reflect altered circadian function, we examined re-entrainment of central and peripheral oscillators from young and old PER2::luciferase mice. As in previous studies, locomotor activity rhythms in older mice required more days to re-entrain following a shift than younger mice. At the tissue level, effects of age on baseline entrainment were evident, with older mice displaying earlier phases for the majority of peripheral oscillators studied and later phases for cells within most SCN subregions. Following a 6 h advance of the light:dark cycle, old mice displayed slower rates of re-entrainment for peripheral tissues but a larger, more rapid SCN response compared to younger mice. Thus, aging alters the circadian timing system in a manner that differentially affects the re-entrainment responses of central and peripheral circadian clocks. This pattern of results suggests that a major consequence of aging is a decrease in pacemaker amplitude, which would slow re-entrainment of peripheral oscillators and reduce SCN resistance to external perturbation.


Assuntos
Envelhecimento/fisiologia , Sistema Nervoso Central/fisiologia , Ritmo Circadiano/fisiologia , Sistema Nervoso Periférico/fisiologia , Animais , Comportamento Animal/fisiologia , Relógios Biológicos/fisiologia , Encéfalo/fisiologia , Interpretação Estatística de Dados , Processamento de Imagem Assistida por Computador , Síndrome do Jet Lag/fisiopatologia , Luciferases/genética , Luciferases/fisiologia , Luminescência , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Atividade Motora/fisiologia , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/fisiologia , Núcleo Supraquiasmático/fisiologia , Técnicas de Cultura de Tecidos
11.
J Pharmacol Exp Ther ; 340(3): 698-705, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22171092

RESUMO

Comprehensive gene expression profiling in mice in response to the inhalation of sevoflurane has revealed that circadian clock gene expression is affected strongly in the liver, heart, lung, and kidney, in this order, but moderately in the spleen and slightly in the brain. Therefore, we examined whether the administration of general anesthetics at different times of the day induces phase shifts of the liver clock in Per2::Luciferase knockin mice. One to 4 days of intraperitoneal injection of 2,2,2-tribromoethanol (240 mg/kg, anesthetic time 60 min) or 2,2,2-trichloroethanol (240 mg/kg, 60 min), common anesthetics in veterinary surgery, caused phase delays when injected during the daytime and phase advances when injected during the nighttime. Inhalation administration of isoflurane for 30 or 60 min during the daytime did not induce a phase delay. Injection of propofol (300 mg/kg, 17 min) during the daytime induced an insignificant phase delay of the Per2 bioluminescence rhythm. Injection of 2,2,2-tribromoethanol did not induce a phase shift in the suprachiasmatic nucleus, the main oscillator, or in behavioral locomotor rhythms, suggesting that 2,2,2-tribromoethanol induced phase shifts of the liver clock independent of the main suprachiasmatic clock. The expression of clock genes, such as Bmal1 and Clock, in mouse liver was decreased strongly 1 and 4 h after a single injection of 2,2,2-tribromoethanol. These results demonstrate that 2,2,2-tribromoethanol or 2,2,2-trichloroethanol produce phase shifts of the peripheral clock, independent of anesthetic activity. These anesthetics may cause circadian rhythm disorders in peripheral organs when administered as general anesthetics several times during the day.


Assuntos
Anestésicos/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Etanol/análogos & derivados , Fígado/efeitos dos fármacos , Luciferases/fisiologia , Proteínas Circadianas Period/fisiologia , Animais , Relação Dose-Resposta a Droga , Etanol/farmacologia , Fígado/metabolismo , Medições Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Proteínas Circadianas Period/genética
12.
Am J Physiol Endocrinol Metab ; 300(5): E877-85, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21343542

RESUMO

The transcription factor HIF-1α activity is increased in adipose tissue to contribute to chronic inflammation in obesity. However, its upstream and downstream events remain to be characterized in adipose tissue in obesity. We addressed this issue by investigating adipocyte HIF-1α activity in response to obesity-associated factors, such as adipogenesis, insulin, and hypoxia. In adipose tissue, both HIF-1α mRNA and protein were increased by obesity. The underlying mechanism was investigated in 3T3-L1 adipocytes. HIF-1α mRNA and protein were augmented by adipocyte differentiation. In differentiated adipocytes, insulin further enhanced HIF-1α in both levels. Hypoxia enhanced only HIF-1α protein, not mRNA. PI3K and mTOR activities are required for the HIF-1α expression. Function of HIF-1α protein was investigated in the regulation of VEGF gene transcription. ChIP assay shows that HIF-1α binds to the proximal hypoxia response element in the VEGF gene promoter, and its function is inhibited by a corepressor composed of HDAC3 and SMRT. These observations suggest that of the three obesity-associated factors, all of them are able to augment HIF-1α protein levels, but only two (adipogenesis and insulin) are able to enhance HIF-1α mRNA activity. Adipose tissue HIF-1α activity is influenced by multiple signals, including adipogenesis, insulin, and hypoxia in obesity. The transcriptional activity of HIF-1α is inhibited by HDAC3-SMRT corepressor in the VEGF gene promoter.


Assuntos
Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Hipoglicemiantes/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/fisiopatologia , Insulina/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adipócitos/metabolismo , Adipócitos/fisiologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular , Imunoprecipitação da Cromatina , Histona Desacetilases/biossíntese , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Luciferases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Correpressor 2 de Receptor Nuclear/biossíntese , Correpressor 2 de Receptor Nuclear/genética , Fosfatidilinositol 3-Quinases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/metabolismo
13.
J Immunol ; 178(11): 7006-17, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17513750

RESUMO

The proinflammatory capacities of the skin and the presence of high numbers of resident dendritic cells (DCs) constitute an ideal microenvironment for successful immunizations. Regardless of the ability of DCs to respond to local inflammatory signals in an immunostimulatory fashion, the immune functions of skin-resident DCs remain controversial, and epidermal Langerhans cells (LCs) have been referred to recently as anti-inflammatory/protolerogenic APCs. Substance P (SP), released by skin nerve fibers, is a potent proinflammatory neuropeptide that favors development of skin-associated cellular immunity. SP exerts its proinflammatory functions by binding with high affinity to the neurokinin 1 receptor (NK1R). In this study, we tested whether signaling skin cells via the NK1R promotes humoral and cellular immunity during skin genetic immunizations. We used the gene gun to deliver transgenic (tg) Ag to the skin of C57BL/6 mice and the selective NK1R agonist [Sar(9)Met (O(2)) (11)]-SP as a potential proinflammatory Th1-biasing adjuvant. Our strategy expressed tg Ag exclusively in the epidermis and induced a preferential migration of activated LCs to skin-draining lymph nodes. Local administration of the NK1R agonist during skin genetic immunizations increased significantly the expression of tg Ag by a mechanism involving the translocation of NF-kappaB into the nuclei of cutaneous DCs homing to skin-draining lymph nodes. Importantly, our immunization approach resulted in Th1 and T cytotoxic (CTL)-1 bias of effector T cells that supported cellular and Ab-mediated immune responses. We demonstrate that signaling skin cells via the NK1R provides the adjuvant effect which favors the immunostimulatory functions of LCs.


Assuntos
Regulação da Expressão Gênica/imunologia , Células de Langerhans/metabolismo , Receptores da Neurocinina-1/fisiologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Transgenes/genética , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/fisiologia , Sequência de Aminoácidos , Animais , Biolística , Galinhas , Feminino , Vaga-Lumes , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/fisiologia , Luciferases/administração & dosagem , Luciferases/biossíntese , Luciferases/genética , Luciferases/fisiologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ovalbumina/administração & dosagem , Ovalbumina/biossíntese , Ovalbumina/genética , Receptores da Neurocinina-1/agonistas , Transdução de Sinais/genética , Pele/imunologia , Pele/metabolismo , Pele/patologia , Linfócitos T Citotóxicos/metabolismo , Células Th1/metabolismo
14.
Artigo em Inglês | MEDLINE | ID: mdl-18419271

RESUMO

The unicellular circadian clock of Gonyaulax polyedra (now renamed Lingulodinium polyedrum) has provided important insights concerning circadian rhythmicity. Many, perhaps most, of its key systems are circadian-controlled, ranging from bioluminescence and photosynthesis to motility, cell division, and the synthesis of many proteins, favoring the "master clock" concept. But different rhythms may have different free-running periods and different phase angles under different T cycles, observations not easily accommodated in a single oscillator model. Gonyaulax has a feature significantly different from that of other known systems, namely, that clock control of protein synthesis occurs at the translational level. With one mRNA, this involves a protein binding to a 22-nucleotide region in the 3'-untranslated region (3'UTR), but no similar regions have been found in other mRNAs. Pulses of protein synthesis inhibitors cause phase shifts, whereas inhibitors of protein phosphorylation administered chronically cause period changes. In Gonyaulax and other systems, low temperature results in arrhythmicity. A return to a permissive temperature results in a reinitiation of the rhythm, with the phase established by the time of increase, similar to the effect of bright light. Evidence for cellular communication via substance(s) in the medium has been obtained.


Assuntos
Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Dinoflagelados/genética , Dinoflagelados/fisiologia , Animais , Comunicação Celular , Temperatura Baixa , Regulação da Expressão Gênica , Genes de Protozoários , História do Século XX , História do Século XXI , Luciferases/genética , Luciferases/história , Luciferases/fisiologia , Biossíntese de Proteínas
15.
Genes Cells ; 11(12): 1381-92, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17121545

RESUMO

Circadian systems coordinate endogenous events with external signals. In mammals, hormone-clock feedbacks are a well-known integration system. Here, we investigated phytohormone effects on plant-circadian rhythms via the promoter:luciferase system. We report that many hormones control specific features of the plant-circadian system, and do so in distinct ways. In particular, cytokinins delay circadian phase, auxins regulate circadian amplitude and clock precision, and brassinosteroid and abscisic acid modulate circadian periodicity. We confirmed the pharmacology in hormone synthesis and perception mutants, as rhythmic expression is predictably altered in an array of hormone-related mutants. We genetically dissected one mechanism that integrates hormone signals into the clock, and showed that the hormone-activated ARABIDOPSIS RESPONSE REGULATOR 4 and the photoreceptor phytochrome B are elements in the input of the cytokinin signal to circadian phase. Furthermore, molecular-expression targets of this signal were found. Collectively, we found that plants have multiple input/output feedbacks, implying that many hormones can function on the circadian system to adjust the clock to external signals to properly maintain the clock system.


Assuntos
Relógios Biológicos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Biológicos/genética , Relógios Biológicos/fisiologia , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Genes Reporter , Luciferases/genética , Luciferases/fisiologia , Medições Luminescentes , Modelos Biológicos , Fitocromo B/genética , Fitocromo B/metabolismo , Fenômenos Fisiológicos Vegetais , Regiões Promotoras Genéticas , Plântula/metabolismo
16.
Biol Reprod ; 74(6): 999-1006, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16495478

RESUMO

Growth differentiation factor 9 (GDF9) is preferentially expressed in oocytes and is essential for female fertility. To identify regulatory elements that confer high-level expression of GDF9 in the ovary but repression in other tissues, we generated transgenic mice in which regions of the Gdf9 locus were fused to reporter genes. Two transgenes (-10.7/+5.6mGdf9-GFP) and (-3.3/+5.6mGdf9-GFP) that contained sequences either 10.7 or 3.3 kb upstream and 5.6 kb downstream of the Gdf9 initiation codon demonstrated expression specifically in oocytes, thereby mimicking endogenous Gdf9 expression. In contrast, transgenes -10.7mGdf9-Luc and -3.3mGdf9-Luc, which lacked the downstream 5.6-kb region, demonstrated reporter expression not only in oocytes but also high expression in male germ cells. This suggests that the downstream 5.6-kb sequence contains a testis-specific repressor element and that 3.3 kb of 5'-flanking sequence contains all the cis-acting elements for directing high expression of Gdf9 to female (and male) germ cells. To define sequences responsible for oocyte expression of Gdf9, we analyzed sequences of Gdf9 genes from 16 mammalian species. The approximately 400 proximal base pairs upstream of these Gdf9 genes are highly conserved and contain a perfectly conserved E-box (CAGCTG) sequence. When this 400-bp region was placed upstream of a luciferase reporter (-0.4mGdf9-Luc), oocyte-specific expression was observed. However, a similar transgene construct (-0.4MUT-mGdf9-Luc) with a mutation in the E-box abolished oocyte expression. Likewise, the presence of an E-box mutation in a longer construct (-3.3MUT-mGdf9-Luc) abolished expression in the ovary but not in the testis. These observations indicate that the E-box is a key regulatory sequence for Gdf9 expression in the ovary.


Assuntos
Elementos E-Box/fisiologia , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Oócitos/metabolismo , Animais , Sequência de Bases , Proteína Morfogenética Óssea 15 , DNA/análise , DNA/genética , Elementos E-Box/genética , Feminino , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Genes Reporter/fisiologia , Fator 9 de Diferenciação de Crescimento , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Luciferases/genética , Luciferases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação/genética , Ovário/metabolismo , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Testículo/metabolismo
17.
Mol Cells ; 20(1): 142-50, 2005 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-16258253

RESUMO

Members of the multifunctional Cyp family have been isolated from a wide range of organisms. However, few functional studies have been performed on the role of these proteins as chaperones in red alga. For studying the function of cDNA GjCyp-1 isolated from the red alga (Griffithsia japonica), we expressed and purified a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus in Escherichia coli. An expressed fusion protein, H6GjCyp-1 maintained the stability of E. coli proteins up to 50 degrees C. For a functional bioassay for recombinant H6GjCyp-1, the viability of E. coli cells overexpressing H6GjCyp-1 was compared with that of cells not expressing H6GjCyp-1 at 50 degrees C. After high temperature treatment for 1 h, E. coli overexpressing H6GjCyp-1 survived about three times longer than E. coli lacking H6GjCyp-1. Measurement of the light scattering of luciferase (luc) showed that GjCyp-1 prevents the aggregation of luc during mild heat stress and that the thermoprotective activity of GjCyp-1 is blocked by cyclosporin A (CsA), an inhibitor of Cyps. Furthermore, the Cyp-CsA complex inhibited the growth of E. coli under normal conditions. The results of the GjCyp-1 bioassays as well as in vitro studies strongly suggest that Cyp confers thermotolerance to E. coli.


Assuntos
Ciclofilinas/fisiologia , Ciclosporina/farmacologia , Rodófitas/fisiologia , Clonagem Molecular , Ciclofilinas/química , Ciclofilinas/genética , Ciclosporina/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/fisiologia , Vetores Genéticos , Histidina/química , Histidina/genética , Luciferases/fisiologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oligopeptídeos/química , Oligopeptídeos/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Rodófitas/efeitos dos fármacos , Rodófitas/genética , Temperatura , Fatores de Tempo
18.
Biochem Biophys Res Commun ; 338(1): 250-3, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16165097

RESUMO

The bioluminescence system in the "firefly squid," Watasenia scintillans, is described. The light-emitting components consist of luciferin (coelenterazine disulfate), a membrane-bound luciferase, ATP, Mg2+, and molecular oxygen. A hypothetical scheme is proposed for the light-emitting reaction.


Assuntos
Decapodiformes/fisiologia , Luminescência , Oxigênio/química , Animais , Decapodiformes/enzimologia , Luciferases/química , Luciferases/fisiologia , Oxigênio/fisiologia
19.
J Immunol ; 174(12): 8125-34, 2005 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15944321

RESUMO

Acute phase serum amyloid A proteins (A-SAAs) are multifunctional apolipoproteins produced in large amounts during the acute phase of an inflammation and also during the development of chronic inflammatory diseases. In this study we present a Saa1-luc transgenic mouse model in which SAA1 gene expression can be monitored by measuring luciferase activity using a noninvasive imaging system. When challenged with LPS, TNF-alpha, or IL-1beta, in vivo imaging of Saa1-luc mice showed a 1000- to 3000-fold induction of luciferase activity in the hepatic region that peaked 4-7 h after treatment. The induction of liver luciferase expression was consistent with an increase in SAA1 mRNA in the liver and a dramatic elevation of the serum SAA1 concentration. Ex vivo analyses revealed luciferase induction in many tissues, ranging from several-fold (brain) to >5000-fold (liver) after LPS or TNF-alpha treatment. Pretreatment of mice with the proteasome inhibitor bortezomib significantly suppressed LPS-induced SAA1 expression. These results suggested that proteasome inhibition, perhaps through the NF-kappaB signaling pathway, may regulate SAA1 expression. During the development of acute arthritis triggered by intra-articular administration of zymosan, SAA1 expression was induced both locally at the knee joint and systemically in the liver, and the induction was significantly suppressed by bortezomib. Induction of SAA1 expression was also demonstrated during contact hypersensitivity induced by topical application of oxazolone. These results suggest that both local and systemic induction of A-SAA occur during inflammation and may contribute to the pathogenesis of chronic inflammatory diseases associated with amyloid deposition.


Assuntos
Artrite Experimental/genética , Dermatite de Contato/genética , Luciferases/genética , Inibidores de Proteassoma , Sepse/enzimologia , Sepse/genética , Proteína Amiloide A Sérica/genética , Doença Aguda , Animais , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Ácidos Borônicos/antagonistas & inibidores , Ácidos Borônicos/farmacologia , Bortezomib , Dermatite de Contato/enzimologia , Dermatite de Contato/patologia , Modelos Animais de Doenças , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , Feminino , Vetores Genéticos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Luciferases/antagonistas & inibidores , Luciferases/biossíntese , Luciferases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Pirazinas/antagonistas & inibidores , Pirazinas/farmacologia , Sepse/patologia , Proteína Amiloide A Sérica/antagonistas & inibidores , Proteína Amiloide A Sérica/biossíntese , Fator de Necrose Tumoral alfa/farmacologia
20.
Patol. apar. locomot. Fund. Mapfre Med ; 3(2): 101-110, abr.-jun. 2005. ilus, tab, graf
Artigo em Es | IBECS | ID: ibc-047414

RESUMO

El cartílago articular es el tejido encargado de disminuirla fricción entre las superficies articulares durante el movimiento.Su limitada capacidad de regeneración hace quelas lesiones osteocondrales posean un mal pronóstico ypuedan acabar generando una artrosis en la articulación.La terapia génica para este tipo de patologías resulta muyprometedora, puesto que actualmente, no hay ningún procedimientocapaz de restablecer el tejido dañado. En nuestroestudio hemos puesto a punto un modelo de transferenciagénica a los tejidos de la articulación de la rodillade rata, inyectando intraarticularmente vectores derivadosde virus adeno-asociados, capaces de inducir la expresiónde luciferasa. Los resultados muestran cómo la inducciónde la expresión resulta significativa a partir de los dos mesesde evolución en todos los tejidos articulares (cartílagoarticular, menisco, membrana sinovial y hueso subcondral).La presencia de daño, tanto de tipo mecánico comoautoinmune (artritis inducida por colágeno) no modifica laexpresión de proteína por parte del vector


Articular cartilage is a tissue that decreases friction onarticular surfaces during movement. Its limited regenerativecapacity makes osteocondral lessions to extend andgenerate osteoarthritis in the joint. Gene therapy is apromising alternative to these patologies, since there areno proceedings actually, that restablishes damaged tissue.In our study, we have developed a model of gene transferto articular tissues injecting intraarticularly an adeno-associatedderived vector that induces expression of luciferase.Results obtained show a significant induction of proteinexpression two months after injecting vectors, in all articulartissues (articular cartilage, meniscus, synovium andsubcondral bone). The presence of lessions, mechanic orautoimmune (collagen induced arthritis) do not modify theexpression of protein induced by the vector


Assuntos
Ratos , Animais , Osteoartrite do Joelho/etiologia , Cartilagem Articular/fisiopatologia , Doenças das Cartilagens/terapia , Recombinação Genética/genética , Dependovirus , Luciferases/fisiologia
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