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1.
J Proteome Res ; 18(8): 3052-3066, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31192604

RESUMO

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree (Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli, was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.


Assuntos
Alérgenos/genética , Antígenos de Plantas/genética , Ciclofilinas/genética , Ciclofilinas/imunologia , Proteoma/genética , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Sequência de Aminoácidos/genética , Animais , Criança , Cromatografia Líquida , Reações Cruzadas , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Olea/efeitos adversos , Olea/genética , Olea/imunologia , Pólen/efeitos adversos , Pólen/genética , Pólen/imunologia , Proteoma/imunologia , Proteômica , Espectrometria de Massas em Tandem
2.
Biofactors ; 45(1): 85-96, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30496631

RESUMO

Rhein, a monomeric anthraquinone obtained from the plant herb species Polygonum multiflorum and P. cuspidatum, has been proposed to have anticancer activity. This activity has been suggested to be associated with mitochondrial injury due to the induction of mitochondrial permeability transition pore (mPTP) opening. In this study, the effects of 5-80 µM rhein on cell viability, half-maximal inhibitory concentration (IC50 value), resistance index, and apoptosis were assessed in the liver cancer cell lines SMMC-7721 and SMMC-7721/DOX (doxorubicin-resistant cells). Rhein (10-80 µM) significantly reduced the viability of both cell lines; 20 µM rhein significantly increased sensitivity to DOX and increased apoptosis in SMMC-7721 cells, but reversed resistance to DOX by 7.24-fold in SMMC-7721/DOX cells. Treatment with rhein increased accumulation of DOX in SMMC-7721/DOX cells, inhibited mitochondrial energy metabolism, decreased cellular ATP, and ADP levels, and altered the ratio of ATP to ADP. These effects may result from the binding of rhein with voltage-dependent ion channels (VDACs), adenine nucleotide translocase (ANT), and cyclophilin D, affecting their function and leading to the inhibition of ATP transport by VDACs and ANT. ATP synthesis was greatly reduced and mitochondrial inner membrane potential decreased. Together, these results indicate that rhein could reverse drug resistance in SMMC-7721/DOX cells by inhibiting energy metabolism and inducing mPTP opening. © 2018 BioFactors, 45(1):85-96, 2019.


Assuntos
Antraquinonas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Antraquinonas/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclofilinas/genética , Ciclofilinas/metabolismo , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Fallopia japonica/química , Fallopia multiflora/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Extratos Vegetais/química , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismo
3.
J Pineal Res ; 65(3): e12503, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29770487

RESUMO

The molecular features of necroptosis in cardiac ischemia-reperfusion (IR) injury have been extensively explored. However, there have been no studies investigating the physiological regulatory mechanisms of melatonin acting on necroptosis in cardiac IR injury. This study was designed to determine the role of necroptosis in microvascular IR injury, and investigate the contribution of melatonin in repressing necroptosis and preventing IR-mediated endothelial system collapse. Our results demonstrated that Ripk3 was primarily activated by IR injury and consequently aggravated endothelial necroptosis, microvessel barrier dysfunction, capillary hyperpermeability, the inflammation response, microcirculatory vasospasms, and microvascular perfusion defects. However, administration of melatonin prevented Ripk3 activation and provided a pro-survival advantage for the endothelial system in the context of cardiac IR injury, similar to the results obtained via genetic ablation of Ripk3. Functional investigations clearly illustrated that activated Ripk3 upregulated PGAM5 expression, and the latter increased CypD phosphorylation, which obligated endothelial cells to undergo necroptosis via augmenting mPTP (mitochondrial permeability transition pore) opening. Interestingly, melatonin supplementation suppressed mPTP opening and interrupted endothelial necroptosis via blocking the Ripk3-PGAM5-CypD signal pathways. Taken together, our studies identified the Ripk3-PGAM5-CypD-mPTP axis as a new pathway responsible for reperfusion-mediated microvascular damage via initiating endothelial necroptosis. In contrast, melatonin treatment inhibited the Ripk3-PGAM5-CypD-mPTP cascade and thus reduced cellular necroptosis, conferring a protective advantage to the endothelial system in IR stress. These findings establish a new paradigm in microvascular IR injury and update the concept for cell death management handled by melatonin under the burden of reperfusion attack.


Assuntos
Vasos Coronários/metabolismo , Ciclofilinas/metabolismo , Melatonina/farmacologia , Microvasos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fosfoproteínas Fosfatases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Vasos Coronários/patologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Camundongos , Camundongos Knockout , Microvasos/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Fosfoproteínas Fosfatases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
4.
Sci Rep ; 6: 38934, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982058

RESUMO

Since about 30% of all human cancers contain mutationally activated Ras, down regulating the over-activation of Ras/MAPK pathway represents a viable approach for treating cancers. Over-activation of Ras/MAPK pathway is accompanied by accumulation of reactive oxygen species (ROS). One approach for developing anti-cancer drugs is to target ROS production and their accumulation. To test this idea, we have employed C. elegans of let-60 (gf) mutant, which contain over-activated let-60 (the homolog of mammalian ras) and exhibit tumor-like symptom of multivulva phenotype, to determine whether anti-oxidants can affect their tumor-like phenotype. Specifically we studied the effect of Shengmai formula (SM), a traditional Chinese medicine that has strong anti-oxidant activity, on the physiology of let-60 (gf) mutants. Unexpectedly, we found that SM treatment led to the opening of mitochondrial permeability transition pore by regulating cyclophilin D and then triggered oxidative stress and related signaling pathway activation, including p53, JNK, and p38/MAPK pathways. Finally, SM induced mitochondrial pathway of apoptosis and inhibited the tumor-like symptom of the multivulva phenotype of let-60(gf) mutants. Our results provide evidences to support that SM act as a pro-oxidant agent and could serve as a potential drug candidate for combating over-activated Ras-related cancer.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Ciclofilinas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Combinação de Medicamentos , Sistema de Sinalização das MAP Quinases/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo
5.
Biochem Biophys Res Commun ; 452(3): 768-74, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25201730

RESUMO

Lung cancer is a major cause of cancer-related mortality in the United States and around the world. Due to the pre-existing or acquired chemo-resistance, the current standard chemotherapy regimens only show moderate activity against lung cancer. In the current study, we explored the potential anti-lung cancer activity of cinobufotalin in vivo and in vitro, and studied the underlying mechanisms. We demonstrated that cinobufotalin displayed considerable cytotoxicity against lung cancer cells (A549, H460 and HTB-58 lines) without inducing significant cell apoptosis. Our data suggest that mitochondrial protein cyclophilin D (Cyp-D)-dependent mitochondrial permeability transition pore (mPTP) opening mediates cinobufotalin-induced non-apoptotic death of lung cancer cells. The Cyp-D inhibitor cyclosporine A (CsA), the mPTP blocker sanglifehrin A (SfA), and Cyp-D shRNA-silencing significantly inhibited cinobufotalin-induced mitochondrial membrane potential (MMP) reduction and A549 cell death (but not apoptosis). Using a mice xenograft model, we found that cinobufotalin inhibited A549 lung cancer cell growth in vivo. Thus, cinobufotalin mainly induces Cyp-D-dependent non-apoptotic death in cultured lung cancer cells. The results of this study suggest that cinobufotalin might be further investigated as a novel anti-lung cancer agent.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Ciclofilinas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Transporte da Membrana Mitocondrial/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Lactonas/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Compostos de Espiro/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cell Death Dis ; 4: e601, 2013 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-23598413

RESUMO

Oroxylin A is a major active component of the Chinese traditional medicinal plant Scutellaria baicalensis Georgi, which has been reported as a potential anticancer drug. We demonstrated that, Oroxylin A inhibited the glycolysis and the binding of hexokinase II (HK II) with mitochondria in human breast carcinoma cell lines, which was dependent on sirtuin-3 (SIRT3). The level of SIRT3 in mitochondria was increased by Oroxylin A. Then SIRT3 deacetylated cyclophilin D, diminished its peptidyl-prolyl cis-trans isomerase activity and induced its dissociation from the adenine nucleotide translocator. Finally, SIRT3-induced inactivation of cyclophilin D resulted in the detachment of mitochondrial HK II and the inhibition of glycolysis. These results have important implications for the metabolism reprogramming effect and the susceptibility to Oroxylin A-induced mitochondrial cytotoxicity through the regulation of SIRT3 in breast carcinoma.


Assuntos
Ciclofilinas/metabolismo , Flavonoides/farmacologia , Glicólise/efeitos dos fármacos , Hexoquinase/metabolismo , Mitocôndrias/efeitos dos fármacos , Sirtuína 3/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Feminino , Flavonoides/química , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Estresse Oxidativo , Raízes de Plantas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Scutellaria/química , Sirtuína 3/antagonistas & inibidores , Sirtuína 3/genética
7.
Am J Physiol Renal Physiol ; 301(1): F134-50, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21490135

RESUMO

Mitochondrial matrix cyclophilin D (CyPD) is known to promote development of the mitochondrial permeability transition (MPT). Kidney proximal tubule cells are especially prone to deleterious effects of mitochondrial damage because of their dependence on oxidative mitochondrial metabolism for ATP production. To clarify the role of CyPD and the MPT in proximal tubule injury during ischemia-reperfusion (I/R) and hypoxia-reoxygenation (H/R), we assessed freshly isolated tubules and in vivo injury in wild-type (WT) and Ppif(-/-) CyPD-null mice. Isolated mouse tubules developed a sustained, nonesterified fatty acid-mediated energetic deficit after H/R in vitro that could be substantially reversed by delipidated albumin and supplemental citric acid cycle substrates but was not modified by the absence of CyPD. Susceptibility of WT and Ppif(-/-) tubules to the MPT was increased by H/R but was less in normoxic and H/R Ppif(-/-) than WT tubules. Correction of the energetic deficit that developed during H/R strongly increased resistance to the MPT. Ppif(-/-) mice were resistant to I/R injury in vivo spanning a wide range of severity. The data clarify involvement of the MPT in oxygen deprivation-induced tubule cell injury by showing that the MPT does not contribute to the initial bioenergetic deficit produced by H/R but the deficit predisposes to subsequent development of the MPT, which contributes pathogenically to kidney I/R injury in vivo.


Assuntos
Ciclofilinas/fisiologia , Hipóxia/patologia , Isquemia/patologia , Túbulos Renais Proximais/fisiologia , Mitocôndrias/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/enzimologia , Membrana Celular/fisiologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ácidos Graxos não Esterificados/metabolismo , Genótipo , Técnicas In Vitro , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/patologia , L-Lactato Desidrogenase/metabolismo , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Permeabilidade , Circulação Renal/fisiologia , Espalhamento de Radiação
8.
Ophthalmic Res ; 45(2): 65-72, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20714194

RESUMO

AIMS: Deduce whether the isoflavone genistein blunts the effect of ischaemia to the retina. METHODS: Ischaemia was induced in rats by raising the intraocular pressure (120 mm Hg) for 50 min. Genistein (10 mg/kg) was injected intraperitoneally 1 h before and after ischaemia. Seven days after ischaemia, the level of mRNAs for neurofilament light (NF-L), caspase 3, caspase 8, glial fibrillary acidic protein (GFAP), poly-ADP ribose polymerase (PARP), Thy-1 and proteins (GFAP, NF-L, PARP) in whole retinas were determined. NF-L and tubulin proteins in optic nerves were also determined. Retinas were also processed for the localization of choline acetyltransferase (ChAT) and GFAP immunoreactivities. RESULTS: Ischaemia caused a significant reduction in ganglion cell proteins in the optic nerve (NF-L and tubulin) and retina (NF-L). Retinal Thy-1 (mRNA and protein) and NF-L (mRNA) were also reduced while mRNAs of caspase 3, caspase 8, PARP and GFAP (also protein) were increased. Changes in the mRNAs and proteins induced by ischaemia were significantly blunted by genistein with the exception of the increase in GFAP and PARP protein/mRNA levels. Ischaemia-induced changes in the localization of ChAT were also clearly attenuated by genistein treatment. CONCLUSIONS: Genistein blunts most of the damaging effects caused to the retina by ischaemia.


Assuntos
Genisteína/uso terapêutico , Pressão Intraocular , Fitoestrógenos/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Doenças Retinianas/prevenção & controle , Animais , Caspase 3/genética , Caspase 8/genética , Ciclofilinas/genética , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/metabolismo , Injeções Intraperitoneais , Proteínas de Neurofilamentos/genética , Hipertensão Ocular/complicações , Hipertensão Ocular/genética , Poli Adenosina Difosfato Ribose/genética , Proteínas/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/genética , Doenças Retinianas/etiologia , Doenças Retinianas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Thy-1/genética
9.
J Biol Chem ; 286(8): 6345-53, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21173147

RESUMO

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.


Assuntos
Encéfalo/enzimologia , Cálcio/metabolismo , Ciclofilinas/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Astrócitos/citologia , Astrócitos/enzimologia , Encéfalo/citologia , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Transporte de Elétrons/fisiologia , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/enzimologia
10.
J Anim Sci ; 86(9): 2296-309, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18441070

RESUMO

Two experiments were conducted to compare performance and metabolic responses of beef females consuming low-quality forages and offered an energy supplement based on fibrous byproducts daily (S7) or 3 times per week (S3) at similar weekly rates. In Exp. 1, BW gain, reproductive performance, mRNA expression of hepatic and skeletal muscle genes associated with nutritional metabolism and growth, and concentrations of blood urea nitrogen (BUN), plasma glucose, insulin, and IGF-I were assessed in 56 Brahman x Angus heifers supplemented at a daily rate of 1.0% of BW. Mean BW gain was greater (P = 0.03) for S7 compared with S3 heifers. Treatment x sampling day interactions were detected (P < 0.01) for all blood measurements. Heifers provided S7 had less daily variation in concentrations of BUN, glucose, and insulin, and frequently had greater (P < 0.05) concentrations of IGF-I compared with S3 heifers. Expression of liver IGF-I mRNA was greater (P = 0.04) for S7 heifers compared with S3 heifers. Treatment x day interactions were detected (P

Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Bovinos/fisiologia , Suplementos Nutricionais , Ingestão de Energia/fisiologia , Reprodução/fisiologia , Animais , Glicemia/metabolismo , Nitrogênio da Ureia Sanguínea , Peso Corporal/fisiologia , Bovinos/genética , Bovinos/metabolismo , Ciclofilinas/química , Ciclofilinas/genética , Feminino , Insulina/sangue , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/genética , Fígado/fisiologia , Músculo Esquelético/fisiologia , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Gravidez , Progesterona/sangue , Piruvato Carboxilase/química , Piruvato Carboxilase/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária
11.
Physiol Plant ; 131(3): 387-98, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18251878

RESUMO

The abundance of a single domain cyclophilin (CyP), designated as SsCyP, was investigated in Solanum sogarandinum and Solanum tuberosum plants during development and in response to various environmental constraints. We show that under control conditions, SsCyP is distributed throughout the plant but in an organ-specific manner. In both Solanum species, the highest protein levels are observed in transporting organs and in tubers, and substantial amounts are noticed in open flowers and in stamens. We also show that the SsCyP abundance in leaves strongly decreases with age. In in vitro-grown plantlets of S. sogarandinum, the SsCyP gene is induced by low temperature at the transcript level but not at the protein level, indicating that post-transcriptional mechanisms control SsCyP expression under cold conditions. In in vivo-grown Solanum plants, the organ-dependent SsCyP protein distribution and abundance are not modified by cold, drought, salinity and photooxidative treatments. In contrast, the protein abundance substantially decreases in all organs of Solanum plants subjected to heat shock. We conclude that the SsCyP protein acts mainly during development and does not belong to the group of stress-induced CyPs.


Assuntos
Ciclofilinas/genética , Temperatura Alta , Proteínas de Plantas/genética , Solanaceae/genética , Sequência de Aminoácidos , Northern Blotting , Western Blotting , Cloroplastos/metabolismo , Ciclofilinas/metabolismo , Flores/genética , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Biossíntese de Proteínas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanaceae/metabolismo , Solanum/genética , Solanum/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Transcrição Gênica
12.
Metabolism ; 55(2): 168-74, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16423622

RESUMO

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.


Assuntos
Enzimas/isolamento & purificação , RNA/isolamento & purificação , Retina/química , Animais , Catalase/análise , Catalase/genética , Ciclofilinas/análise , Ciclofilinas/genética , Enzimas/química , Feminino , Glutamato-Cisteína Ligase/análise , Glutamato-Cisteína Ligase/genética , Glutationa Peroxidase/análise , Glutationa Peroxidase/genética , Immunoblotting , Peptidilprolil Isomerase/análise , Peptidilprolil Isomerase/genética , RNA/química , RNA/genética , Ratos , Ratos Wistar , Retina/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/análise , Superóxido Dismutase/genética , Glutationa Peroxidase GPX1
13.
Eur J Biochem ; 271(20): 4084-93, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15479237

RESUMO

Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.


Assuntos
Aminoácidos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Marcação por Isótopo/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Biossíntese de Proteínas/genética , Aminoácidos/química , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Bacillus subtilis/enzimologia , Bacteriófago T7/genética , Bacteriófago lambda/genética , Bovinos , Sistema Livre de Células , Ciclofilina A/análise , Ciclofilina A/biossíntese , Ciclofilina A/química , Ciclofilina A/genética , Ciclofilinas/análise , Ciclofilinas/biossíntese , Ciclofilinas/química , Ciclofilinas/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Humanos , Cinética , Lupinus/enzimologia , Lupinus/genética , Isótopos de Nitrogênio , Paracoccus denitrificans/enzimologia , Peptidilprolil Isomerase , Regiões Promotoras Genéticas , Saccharomyces/enzimologia , Saccharomyces/genética , Proteínas Virais
14.
Biosci Biotechnol Biochem ; 68(3): 508-15, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15056880

RESUMO

Modulation of the activity and content of cytochrome P-450 (CYP) in hepatic microsomes may be important to human health since these enzymes activate and inactivate a wide range of xenobiotics and food components. Regulation of the inducibility of most CYPs involves transcriptional regulation and post-transcriptional mRNA stabilization. We examined in the present study the effect of dietary soy isoflavone (0-300 mg of isoflavone/kg of diet) on the mRNA abundance of rat hepatic CYP1A1, CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A1, CYP3A2 and CYP4A1 by quantitative competitive RT-PCR and real-time monitored RT-PCR. A fermented soy extract containing 155 mg/g of genistein, 127 mg/g of daidzein, and other minor isoflavones was used as the isoflavone source. The dietary soy isoflavone had no affect on the hepatic mRNA abundance of these CYPs. The results by both methods were well matched and indicate that the dietary soy isoflavone did not cause the induction of CYPs by transcriptional step-up regulation or post-transcriptional mRNA stabilization.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Glycine max/química , Isoflavonas/farmacologia , Fígado/enzimologia , Animais , Peso Corporal/efeitos dos fármacos , Ciclofilinas/efeitos dos fármacos , Ciclofilinas/genética , Ciclofilinas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Ingestão de Alimentos/efeitos dos fármacos , Indução Enzimática , Feminino , Indóis/farmacologia , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Isoflavonas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glycine max/metabolismo
15.
Genetics ; 163(3): 1047-60, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12663543

RESUMO

We used a genetic screening methodology, a human cell line bearing a retinoic-acid-responsive enhanced GFP reporter, and a flow sorter to recover dominant modulators of reporter expression. Four inducers and three suppressors that were fused to the C terminus of a protein scaffold for stability were isolated and their mechanisms of action studied. Mutagenesis experiments indicated that six of these dominant agents exerted their effects at the protein level. The single cDNA coding fragment that was isolated comprised the central 64-amino-acid section of human cyclophilin B, which contained its peptidyl-prolyl isomerase domain; this cyclophilin fragment repressed expression of the retinoic-acid-responsive reporter. The remaining clones encoded peptides shorter than 30 amino acids unrelated to known gene open reading frames. Genetic epistasis studies between the strongest inducer, R3, and a dominant-negative mutant of RARalpha suggest that the two factors function in the same pathway. Transcript microarray analyses suggest that R3 induced a subset of the retinoid-responsive genes in melanoma cells. Finally, yeast two-hybrid assays and co-immunoprecipitation studies of human cell extracts identified PAT1 as a protein that interacts with R3.


Assuntos
Testes Genéticos/métodos , Receptores do Ácido Retinoico/genética , Seleção Genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Ciclofilinas/química , Ciclofilinas/genética , DNA Complementar/genética , DNA Mitocondrial/genética , Biblioteca Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Peptidilprolil Isomerase , Mapeamento por Restrição , Transfecção
16.
Physiol Genomics ; 12(2): 163-74, 2003 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12419855

RESUMO

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Interassay variability was 2.3% for raw C(T) values and 34% for the linear value of 2(-C(T)). We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C(T) values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas beta-actin, beta2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for beta2M with more stable expressions for both beta-actin and CYC. We conclude that, using real-time RT-PCR, beta-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.


Assuntos
Creatina/farmacologia , Suplementos Nutricionais , Genes/efeitos dos fármacos , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Actinas/genética , Actinas/metabolismo , Adulto , Análise de Variância , Sistemas Computacionais/estatística & dados numéricos , Creatina/metabolismo , Estudos Cross-Over , Ciclofilinas/biossíntese , Ciclofilinas/genética , Ciclofilinas/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , DNA Complementar/metabolismo , Método Duplo-Cego , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Variação Genética/genética , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Masculino , Músculo Esquelético/metabolismo , Projetos Piloto , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Microglobulina beta-2/biossíntese , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
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