RESUMO
Baculovirus-host interactions are important models for studying the biological control of lepidopteran pests. Research on baculovirus-host interactions has focussed on baculovirus manipulation of cellular signalling pathways, including the extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinases/protein kinase B (PI3K/Akt) signalling pathways. However, the mechanism underlying ERK and PI3K/Akt activation and function in response to baculovirus infection remains poorly understood. Here, we demonstrated that baculovirus activated the Bombyx mori ERK and PI3K/Akt signalling pathways via the B. mori epidermal growth factor receptor (BmEGFR). To further characterize the function of the BmEGFR/ERK signalling pathway in baculovirus replication, we calculated genome-wide changes in kinase-chromatin interactions for ERK after baculovirus infection using chromatin immunoprecipitation followed by high-throughput sequencing. A Gene Ontology analysis showed that virus infection had effects on the biological regulation, cellular process and metabolic process pathways. Moreover, ERK was shown to regulate the transcription of late viral genes. Taken together, our results suggest that baculoviruses manipulate components of the host cell machinery for replication via modulation of the BmEGFR signalling pathway.
Assuntos
Proteínas de Insetos/genética , Nucleopoliedrovírus/fisiologia , Transdução de Sinais/genética , Replicação Viral , Animais , Bombyx , Receptores ErbB , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologiaRESUMO
Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.
Assuntos
Evolução Molecular , Saxitoxina/genética , Canais de Sódio/genética , Tetraodontiformes/genética , Compostos de Trialquitina/metabolismo , Animais , Bases de Dados Genéticas , Filogenia , Especificidade da Espécie , Tetraodontiformes/classificaçãoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is recognized as the hepatic component of the metabolic syndrome. Although NAFLD is a major cause of cirrhosis and cancer of the liver of unknown cause, no established pharmacological treatment for NAFLD has been established yet. It has been reported that leptin treatment improved fatty liver dramatically as well as insulin resistance and hyperphagia in patients with lipodystrophy. However, it is unclear whether leptin improves fatty liver independently of these metabolic improvements. We investigated the liver effect of leptin independently of insulin sensitization and appetite suppression using hepatocyte-specific Pten-deficient (AlbCrePtenff) mouse, a model of severe fatty liver with insulin hypersensitivity. Male AlbCrePtenff mice were infused subcutaneously with leptin (20 ng/g/h) for 2 weeks using osmotic minipumps. Leptin infusion effectively reduced liver weight, liver triglyceride content, and glutamate pyruvate transaminase (GPT) concentrations as well as food intake and body weight without the change of plasma insulin concentration in AlbCrePtenff mice. Pair-feeding also reduced body weight but not liver triglyceride content. Pair feeding reduced α1 and α2 AMP-activated protein kinase (AMPK) activities and PGC1α gene expression in the liver, while leptin infusion unchanged them. The present study clearly demonstrated that leptin improve fatty liver independently of insulin sensitization and suppression of food intake. It was suggested that leptin improves fatty liver by stimulation of ß-oxidation in the liver. The present study might provide a further understanding on the mechanism of metabolic effect of leptin.
Assuntos
Hepatócitos/metabolismo , Insulina/metabolismo , Leptina/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apetite/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Triglicerídeos/metabolismoRESUMO
The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals.
Assuntos
Bombyx/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Bombyx/metabolismo , Linhagem Celular , Proliferação de Células , Reparo do DNA , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Mitomicina/farmacologia , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , UbiquitinaçãoRESUMO
Small RNA-mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA-induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double-stranded RNA (dsRNA)-induced RNAi, whereas Ago3 and Siwi are involved in the Piwi-interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)-mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA-decapping enzyme 2 (Dcp2) protein in the Drosophila processing-bodies (P-bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P-bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.
Assuntos
Proteínas Argonautas/genética , Bombyx/genética , Proteínas de Insetos/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Argonautas/metabolismo , Bombyx/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Imunoprecipitação , Proteínas de Insetos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Biossíntese de Proteínas , Interferência de RNA , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.
Assuntos
Bombyx/embriologia , Bombyx/enzimologia , Embrião não Mamífero/enzimologia , Integrases/metabolismo , Animais , Bombyx/genética , Feminino , Integrases/genética , Mutagênese Insercional/genética , Plasmídeos/genética , Recombinação GenéticaRESUMO
AIMS/HYPOTHESIS: Evidence suggests that telmisartan, an angiotensin II type 1 receptor (AT1) blocker and peroxisome proliferator-activated receptor-gamma partial agonist, has beneficial actions that limit development of the metabolic syndrome and diabetes. However, the role played by AT1 inhibition in metabolic effects elicited by telmisartan remains uncertain. Here we isolated the metabolic effects of telmisartan from AT1 antagonism. METHODS: Male At1a (also known as Agtr1a)-deficient mice were fed a standard diet or 60% high-fat diet; those on high-fat diet were co-administered telmisartan (3 mg kg(-1) day(-1) by oral gavage) or vehicle for 12 weeks. RESULTS: In At1a-null mice, telmisartan prevented high-fat-diet-induced increases in (1) body weight, epididymal and inguinal white adipose tissue weight, adipocyte size and plasma leptin concentration; (2) plasma glucose and insulin concentrations and HOMA index; and (3) liver weight and triacylglycerol content. Insulin tolerance testing also indicated that telmisartan improved the high-fat-diet-induced reduction of glucose-lowering by insulin. CONCLUSIONS/INTERPRETATION: The present findings demonstrate beneficial, AT1-independent effects of the AT1 blocker telmisartan on dietary-induced obesity, insulin resistance and fatty liver in animals.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Benzimidazóis/administração & dosagem , Benzoatos/administração & dosagem , Fígado Gorduroso/tratamento farmacológico , Resistência à Insulina , Obesidade Abdominal/tratamento farmacológico , Receptor Tipo 1 de Angiotensina/fisiologia , Adipócitos/patologia , Tecido Adiposo Branco/patologia , Animais , Glicemia/análise , Tamanho Celular , Dieta Hiperlipídica , Fígado Gorduroso/patologia , Insulina/sangue , Leptina/sangue , Lipídeos/análise , Fígado/química , Fígado/patologia , Masculino , Síndrome Metabólica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/química , Obesidade Abdominal/etiologia , Tamanho do Órgão , PPAR gama/agonistas , Receptor Tipo 1 de Angiotensina/deficiência , Telmisartan , Triglicerídeos/análiseRESUMO
AIMS/HYPOTHESIS: We have previously demonstrated the therapeutic usefulness of leptin in lipoatrophic diabetes and insulin-deficient diabetes in mouse models and could also demonstrate its dramatic effects on lipoatrophic diabetes in humans. The aim of the present study was to explore the therapeutic usefulness of leptin in a mouse model of type 2 diabetes with increased adiposity. METHODS: To generate a mouse model mimicking human type 2 diabetes with increased adiposity, we used a combination of low-dose streptozotocin (STZ, 120 microg/g body weight) and high-fat diet (HFD, 45% of energy as fat). Recombinant mouse leptin was infused chronically (20 ng [g body weight](-1) h(-1)) for 14 days using a mini-osmotic pump. The effects of leptin on food intake, body weight, metabolic variables, tissue triacylglycerol content and AMP-activated protein kinase (AMPK) activity were examined. RESULTS: Low-dose STZ injection led to a substantial reduction of plasma insulin levels and hyperglycaemia. Subsequent HFD feeding increased adiposity and induced insulin resistance and further augmentation of hyperglycaemia. In this model mouse mimicking human type 2 diabetes (STZ/HFD), continuous leptin infusion reduced food intake and body weight and improved glucose and lipid metabolism with enhancement of insulin sensitivity. Leptin also decreased liver and skeletal muscle triacylglycerol content accompanied by an increase of alpha2 AMPK activity in skeletal muscle. Pair-feeding experiments demonstrated that leptin improved glucose and lipid metabolism independently of the food intake reduction. CONCLUSIONS/INTERPRETATION: This study demonstrates the beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity, indicating the possible clinical usefulness of leptin as a new glucose-lowering drug in humans.
Assuntos
Tecido Adiposo/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Gorduras na Dieta/farmacologia , Ingestão de Energia/efeitos dos fármacos , Leptina/uso terapêutico , Estreptozocina/farmacologia , Redução de Peso/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The purpose of the present study is to identify genes that contribute to cell proliferation or differentiation of breast cancers independent of signalling through the oestrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). An oligonucleotide microarray assayed 40 tumour samples from ER(+)/HER2(-), ER(+)/HER2(+), ER(-)/HER2(+), and ER(-)/HER2(-) breast cancer tissues. Quantitative reverse transcriptase PCR detected overexpression of a cell cycle-related transcription factor, E2F-5, in ER-negative breast cancers, and fluorescence in situ hybridisation detected gene amplification of E2F-5 in 5 out of 57 (8.8%) breast cancer samples. No point mutations were found in the DNA-binding or DNA-dimerisation domain of E2F-5. Immunohistochemically, E2F-5-positive cancers correlated with a higher Ki-67 labelling index (59.5%, P=0.001) and higher histological grades (P=0.049). E2F-5-positive cancers were found more frequently in ER(-)/progesterone receptor (PgR)(-)/HER2(-) cancer samples (51.9%, P=0.0049) and in breast cancer samples exhibiting a basal phenotype (56.0%, P=0.0012). Disease-free survival in node-negative patients with E2F-5-positive cancers was shorter than for patients with E2F-5-negative cancers. In conclusion, we identify, for the first time, a population of breast cancer cells that overexpress the cell cycle-related transcription factor, E2F-5. This E2F-5-positive breast cancer subtype was associated with an ER(-)/PgR(-)/HER2(-) status, a basal phenotype, and a worse clinical outcome.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/patologia , Fator de Transcrição E2F5/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Análise Citogenética , Citoplasma/metabolismo , Análise Mutacional de DNA , Fator de Transcrição E2F5/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Prognóstico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Distribuição Tecidual , Regulação para CimaRESUMO
Adipose tissue plays an important role in energy balance and metabolism and is the major target for insulin-sensitizing peroxisome proliferator-activated receptor (PPAR) gamma agonists. The angiotensin II type 1 receptor blocker telmisartan, a partial agonist of PPAR-gamma, has been demonstrated to improve insulin sensitivity. However, there is uncertainty about the sites of its action. Here, we demonstrate that treatment with telmisartan (3 mg/kg p.o.) for 7 weeks decreased plasma glucose levels in oral glucose and insulin tolerance tests and the index of the homeostasis model assessment of insulin resistance in A-ZIP/F-1 transgenic mice, an animal model of lipodystrophy. These effects were accompanied by decreases in circulating triglyceride and fatty acid levels. However, this treatment did not affect body weight and plasma adiponectin, leptin, and corticosterone levels. In A-ZIP/F-1 mouse liver the transcripts encoding PPAR-gamma and its downstream lipogenic genes were highly up-regulated, consistent with increased hepatic triglyceride content and lipid droplet accumulation. Telmisartan reversed these effects and also down-regulated mRNAs encoding gluconeogenic genes. Thus, the present findings are consistent with a novel mode of insulin-sensitizing action of telmisartan, involving an adipose tissue-independent pathway. Telmisartan-elicited down-regulation of hepatic expression of PPAR-gamma-regulated lipogenic genes is associated with amelioration of fatty liver.
Assuntos
Tecido Adiposo/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Insulina/metabolismo , Lipodistrofia/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Animais , Benzimidazóis/administração & dosagem , Benzoatos/administração & dosagem , Glicemia/análise , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Feminino , Teste de Tolerância a Glucose , Resistência à Insulina , Lipodistrofia/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacos , PPAR gama/agonistas , Telmisartan , Fatores de Transcrição/genética , Triglicerídeos/metabolismoRESUMO
We present 4 cases of facet cyst haematoma in the ligamentum flavum of the lumbar spine. All patients presented with a one-to-3-month history of back pain or numbness in the legs, and sudden neurological deterioration. One also developed cauda equina syndrome and another developed radiculopathy. In all cases, magnetic resonance imaging showed a mass with high signal intensity on both T1- and T2-weighted images. Facet arthrography and computed tomography revealed communication between the mass and the neighbouring facet joint. The haematomas were removed en bloc with the ligamentum flavum. They were surrounded by the ligament and contained degenerated and lacerated elastic fibres but no synovial lining cells. Facet cyst haematoma is so-named because of bleeding from tissue adjacent to the facet joint into a pre-existing facet cyst.
Assuntos
Cistos/diagnóstico , Hematoma/diagnóstico , Artropatias/diagnóstico , Ligamento Amarelo , Vértebras Lombares , Articulação Zigapofisária , Idoso de 80 Anos ou mais , Cistos/etiologia , Cistos/cirurgia , Hematoma/etiologia , Hematoma/cirurgia , Humanos , Artropatias/etiologia , Artropatias/cirurgia , Masculino , Pessoa de Meia-IdadeRESUMO
This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.
Assuntos
Metais/metabolismo , Phaeophyceae/química , Polissacarídeos/farmacologia , Alga Marinha/química , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Imuno-Histoquímica , Ratos , Análise EspectralRESUMO
The objectives of this research were to investigate seasonal and spatial variations in (1) sorption of pyrene and its derivatives onto dissolved organic matter (DOM) and (2) fluorescence properties of DOM in Lake Biwa, Japan. In the case of pyrene, sorption coefficient (Kdoc) of Lake Biwa DOM seasonally changed from 1,200 to 3,800 L/kgC. Vertical distribution of Kdoc was affected by thermocline formation in summer, while it was uniformly distributed as a result of vertical mixing in winter. Functional groups affected sorption of pyrene onto Lake Biwa DOM in different manner from that onto Suwannee River fulvic acid. Three-dimensional excitation emission matrices (3D-EEMs) fluorescence spectroscopy was applied to characterize Lake Biwa DOMs and indicated the existence of at least two fluorophores. The two major peaks at Ex230/Em300 and Ex230/Em425 originated from protein-like and fulvic/humic-like substances, respectively. The peak at Ex230/Em300 showed the maximum fluorescence intensity at a depth of 5 m and could be affected by stratification of the water column in summer. On the other hand, the peak at Ex230/Em425 showed similar profiles both in summer and in winter. These results demonstrably showed that sorption of micropollutants and fluorescence properties of Lake Biwa DOMs were seasonally and spatially varied.
Assuntos
Água Doce/química , Pirenos/análise , Estações do Ano , Pirenos/química , Espectrometria de FluorescênciaRESUMO
Gonadotropin-releasing hormone (GnRH) is a ten-amino acid peptide hormone that plays pivotal roles in reproduction in vertebrates and octopus. Recently, six GnRH forms (t-GnRH-3-8) and four GnRH receptor subtypes (Ci-GnRHR-1-4) were identified in the protochordate, Ciona intestinalis. In this study, we show the functional modulation of Ci-GnRHR-1 via heterodimerization with the orphan receptor subtype, Ci-GnRHR-4. The dimerization between Ci-GnRHR-1 and R-4 was detected by co-immunoprecipitation and immunoblot analysis. Binding assays confirmed the binding of t-GnRHs to Ci-GnRHR-1 but not to R-4, and verified no alternation in ligand-binding affinity between Ci-GnRHR-1 homodimer and Ci-GnRHRI&4 heterodimer. The heterodimer was found to stimulate the elevation of intracellular calcium, time-extension of ERK phosphorylation, and up-regulation of cell proliferation, all in a ligand specific manner, compared with the Ci-GnRHR-1 homodimer. In combination, these results indicated that Ci-GnRHR-4 is not an inactive receptor, but a modulatory factor for Ci-GnRHR-1 in C. intestinalis.
Assuntos
Ciona intestinalis/metabolismo , Receptores LHRH/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células , Ciona intestinalis/citologia , Dimerização , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Estrutura Quaternária de Proteína , Receptores LHRH/química , Receptores LHRH/classificação , Transdução de SinaisRESUMO
AIM: The functional significance of the myokines, cytokines and peptides produced and released by muscle cells has not been fully elucidated. The purpose of this study was to identify a myokine with increased secretion levels in muscle cells due to saturated fatty acids and to examine the role of the identified myokine in the regulation of myogenesis. METHODS: Human primary myotubes and mouse C2C12 myotubes were used to identify the myokine; its secretion was stimulated by palmitate loading. The role of the identified myokine in the regulation of the activation, proliferation, differentiation and self-renewal was examined in mouse satellite cells (skeletal muscle stem cells). RESULTS: Palmitate loading promoted the secretion of chemokine (C-X-C motif) ligand 1 (CXCL1) in human primary myotubes, and it also increased CXCL1 gene expression level in C2C12 myotubes in a dose- and time-dependent manner. Palmitate loading increased the production of reactive oxygen species along with the activation of nuclear factor-kappa B (NF-κB) signalling. Pharmacological inhibition of NF-κB signalling attenuated the increase in CXCL1 gene expression induced by palmitate and hydrogen peroxide. Palmitate loading significantly increased CXC receptor 2 gene expression in undifferentiated cells. CXCL1 knockdown attenuated proliferation and myotube formation by satellite cells, with reduced self-renewal. CXCL1 knockdown also significantly decreased the Notch intracellular domain protein level. CONCLUSION: These results suggest that secretion of the myokine CXCL1 is stimulated by saturated fatty acids and that CXCL1 promotes myogenesis from satellite cells to maintain skeletal muscle homeostasis.
Assuntos
Quimiocina CXCL1/metabolismo , Desenvolvimento Muscular/fisiologia , Palmitatos/farmacologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Animais , Humanos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismoRESUMO
Increased activity of intracellular glucocorticoid reactivating enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11beta-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11beta-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C(2) ceramide, bacterial sphingomyelinase, and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11beta-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C(2) ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) beta and its binding to 11beta-HSD1 promoter. Transient knockdown of C/EBPbeta protein by small interfering RNA markedly attenuated the expression of 11beta-HSD1 induced by C(2) ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11beta-HSD1 in preadipocytes by way of C/EBPbeta, thereby highlighting a novel, metabolic stress-related regulation of 11beta-HSD1 in a cell-specific manner.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Adipócitos/metabolismo , Ceramidas/fisiologia , Complexos Multienzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Ceramidas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE) and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with 3 mg/kg body weight of CdCl2 thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity. The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced toxicity.
Assuntos
Cádmio/análise , Cádmio/metabolismo , Córtex Renal/metabolismo , Fígado/metabolismo , Animais , Cádmio/efeitos adversos , Esquema de Medicação , Feminino , Córtex Renal/patologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos ICR , Espectrometria por Raios X , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
A cDNA encoding for a heat shock protein 30 (HSP30) of Aspergillus nidulans and the promoter region of its gene were analyzed for their primary structures. The promoter region had no heat shock element but possessed three inverted repeat sequences. Northern blot hybridization indicated that the expression of the HSP30 gene was high at a normal temperature and was slightly accelerated at elevated temperatures in A. nidulans cells. Although the deduced amino acid sequence of the A. nidulans HSP30 had a domain highly conserved among other small HSPs from different species, it showed a sequence homology of only 42% even in comparison with the most closely related molecular species, Neurospora crassa HSP30. These findings suggest that the present HSP30 belongs to a novel subfamily of low-molecular-weight HSPs.
Assuntos
Aspergillus nidulans/genética , Proteínas de Choque Térmico/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Evidence is presented that the sequence-specific DNA binding of esperamicin is accompanied by structural distortion of the host DNA that depends on hydrophobic interactions. In the first part of this paper, we describe the effects of conformational freedom of DNA on the DNA-cutting efficiency of esperamicin. If conformational distortion of DNA is significantly required for binding of the drug, then the drug should possess a lower binding/cleaving efficiency for conformationally more restricted DNAs. Our results on model DNAs reveal a substantial decrease in the DNA-cleaving efficiency of esperamicin as the conformational freedom of DNA decrease. In the second part we demonstrate that esperamicin binds to DNA with considerably higher affinity in solutions containing organic solvents. This observation indicates that the required distortion of DNA depends on hydrophobic interactions. Molecular origins of the sequence-specific binding are also discussed on the basis of all available data.
Assuntos
Aminoglicosídeos , Antibacterianos/metabolismo , DNA/metabolismo , Conformação de Ácido Nucleico , Sequência de Bases , Enedi-Inos , Dados de Sequência Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Western blot analyses of yolk proteins of the White Leghorn hens showed that an ovalbumin-like molecule was included in day 16 eggs but not in the ovarian follicles, and very little in newly deposited eggs. Northern hybridization, as well as in vitro translation, of poly(A)+ RNAs prepared from the yolk sac membranes of developing embryos gave no signal for ovalbumin messages. These results imply that the present ovalbumin-like protein of yolk has its origin in egg white, not being a de novo synthesis product in the yolk sac membranes.