RESUMO
Dendritic cells (DCs) play a crucial role as central orchestrators of immune system response in atherosclerosis initiation and progression. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the immune maturation of DCs, but the underlying mechanisms remain unclear. We isolated mouse bone marrow progenitors and stimulated them with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce immature DCs. We then treated DCs with oxidized low-density lipoprotein (oxLDL) to induce maturation. LOX-1 siRNA was used to investigate the modulation of LOX-1 on the development of DCs and the underlying signal pathways. CD11c-positive DCs were successfully derived from mouse bone marrow progenitors. OxLDL promoted the expressions of DCs maturation markers and pro-inflammatory cytokines. OxLDL also upregulated LOX-1 expression and activated MAPK/NF-κB pathways. LOX-1 siRNA could attenuate the expression of MAPK/NF-κB pathways and inflammatory cytokines. In conclusion, oxLDL induced the maturation of DCs via LOX-1-mediated MAPK/NF-κB pathway, which contributed to the initiation and progression of atherosclerosis.
Assuntos
Células Dendríticas , Lipoproteínas LDL , Sistema de Sinalização das MAP Quinases , NF-kappa B , Receptores Depuradores Classe E , Animais , CamundongosRESUMO
One lung ventilation (OLV) often results in trauma to the unventilated contralateral lung. This study aims to evaluate the effects of different OLV regimens on the injury of the unventilated contralateral lung to identify the best conditions for OLV. Forty rabbits were divided into five groups: a sham group, OLV group I (fraction of inspired oxygen (FIO2) 1.0, tidal volume (VT) 8mL/kg, respiratory rate (R) 40 breaths/min and inspiratory/expiratory ratio (I:E) 1:2), OLV group II (FIO2=1.0, VT 8mL/kg, R 40 breaths/min, I:E 1:2, and positive end-expiratory pressure (PEEP) 5 cm H2O), OLV group III (FIO2 1.0, VT 6mL/kg, R 40 breaths/min, I:E 1:2 and PEEP 5 cm H2O) and OLV group IV (FIO2 0.8, VT 6mL/kg, R 40 breaths/min, I:E 1:2 and PEEP 5 cm H2O). Animals from all OLV groups received two-lung ventilation (TLV) to establish a baseline, followed by one of the indicated OLV regimens. The rabbits in the sham group were intubated through trachea and ventilated with fresh air. Arterial blood gas samples were collected, lung injury parameters were evaluated, and the concentrations of TNF-α and IL-8 in bronchoalveolar lavage fluid (BALF) and pulmonary surfactant protein A (SPA) in the unventilated lung were also measured. In OLV group I, the unventilated left lung had higher TNF-α, IL-8 and lung injury score but lower SPA than the ventilated right lung. In OLV groups I to III, the concentrations of TNF-α, IL-8 and lung injury score in the left lung decreased but SPA increased. No differences in these parameters between OLV groups III and IV were observed. Strategic ventilation designed for OLV groups III and IV reduced OLV-induced injury of the non-ventilated contralateral lung in rabbits.(AU)
Ventilação pulmonar unilateral (OLV) frequentemente resulta em trauma no pulmão contralateral não ventilado. Este estudo visa avaliar os efeitos de diferentes regimes de OLV sobre a lesão do pulmão contralateral não ventilado para identificar as melhores condições para OLV. Quarenta coelhos foram divididos em cinco grupos: um grupo falso, OLV grupo I (fração de oxigênio inspirado (FIO2) 1.0, volume corrente (VT) 8mL/kg, frequência respiratória (R) 40 respirações/min e relação inspiração/expiração (I:E) 1:2), OLV grupo II (FIO2=1.0, VT 8mL/kg, R 40 respirações/min, I:E 1:2, e pressão positiva expiratória final (PEEP) 5 cm H2O), OLV grupo III (FIO2 1.0, VT 6mL/kg, R 40 respirações/min, I:E 1:2 e PEEP 5 cm H2O) e OLV grupo IV (FIO2 0.8, VT 6mL/kg, R 40 respirações/min, I:E 1:2 e PEEP 5 cm H2O). Os animais de todos os grupos OLV receberam ventilação nos dois pulmões (TLV) para estabelecer uma linha de base, seguida por um dos regimes OLV indicados. Os coelhos do grupo falso foram intubados através da traqueia e ventilados com ar fresco. Amostras de gases no sangue arterial foram coletadas, parâmetros de lesão pulmonar foram avaliados e as concentrações de TNF-α e IL-8 no fluido de lavagem bronco alveolar (BALF) e proteína A do surfactante pulmonar (SPA) no pulmão não ventilado também foram medidas. No grupo OLV I, o pulmão esquerdo não ventilado tinha maior índice de TNF-α, IL-8 e lesão pulmonar, mas menor SPA do que o pulmão direito ventilado. Nos grupos OLV I a III, as concentrações de TNF-α, IL-8 e a pontuação de lesão pulmonar no pulmão esquerdo diminuíram, mas o SPA aumentou. Não foram observadas diferenças nestes parâmetros entre os grupos OLV III e IV. A ventilação estratégica projetada para os grupos OLV III e IV reduziu a lesão induzida por OLV do pulmão contralateral não ventilado em coelhos.(AU)
Assuntos
Animais , Coelhos , Ventilação Pulmonar , Lesão Pulmonar Aguda/complicações , Ventilação Monopulmonar/veterináriaRESUMO
Dendritic cells (DCs) play a crucial role as central orchestrators of immune system response in atherosclerosis initiation and progression. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the immune maturation of DCs, but the underlying mechanisms remain unclear. We isolated mouse bone marrow progenitors and stimulated them with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce immature DCs. We then treated DCs with oxidized low-density lipoprotein (oxLDL) to induce maturation. LOX-1 siRNA was used to investigate the modulation of LOX-1 on the development of DCs and the underlying signal pathways. CD11c-positive DCs were successfully derived from mouse bone marrow progenitors. OxLDL promoted the expressions of DCs maturation markers and pro-inflammatory cytokines. OxLDL also upregulated LOX-1 expression and activated MAPK/NF-κB pathways. LOX-1 siRNA could attenuate the expression of MAPK/NF-κB pathways and inflammatory cytokines. In conclusion, oxLDL induced the maturation of DCs via LOX-1-mediated MAPK/NF-κB pathway, which contributed to the initiation and progression of atherosclerosis.
Assuntos
Animais , Ratos , Células Dendríticas , NF-kappa B , Sistema de Sinalização das MAP Quinases , Receptores Depuradores Classe E , Lipoproteínas LDLRESUMO
Melon (Cucumis melo L.) is an important vegetable crop that ranks second in salt tolerance among the Cucurbitaceae. Previous studies on the two muskmelon cultivars 'Bing XueCui' (BXC) and 'Yu Lu' (YL) revealed that they had different characteristics under salt stress, but the molecular basis underlying their different physiological responses is unclear. Here, we combined a physiological study with a genome-wide transcriptome analysis to understand the molecular basis of genetic variation that responds to salt stress in the melon. BXC performed better under salt stress than YL in terms of biomass and photosynthetic characteristics, because it exhibited less reduction in transpiration rate, net photosynthesis rate, and stomatal conductance under 150-mM NaCl stress than YL. A transcriptome comparison of the leaves of the cultivars revealed that 1171 genes responded to salt stress in BXC while 1487 genes were identified as salt-stress-responsive in YL. A real-time polymerase chain reaction analysis of 12 of the responsive genes revealed that there was a strong, positive correlation with RNA sequencing data. The genes were involved in several pathways, including photosynthesis, the biosynthesis of secondary metabolites, metabolism, and plant hormone signal transduction, and their expression levels differed between the two cultivars in response to salt stress. This study provides a molecular perspective of two melon cultivars in response to salt stress, and its results could be used to investigate the complex molecular mechanisms underlying salt tolerance in the melon.
Assuntos
Cucumis melo/fisiologia , Plantas Tolerantes a Sal/fisiologia , Cucumis melo/genética , Expressão Gênica , Perfilação da Expressão Gênica , Salinidade , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/genética , Estresse Fisiológico/genética , TranscriptomaRESUMO
The dehydrogenase/reductase (SDR family) member 4 (DHRS4) gene is copied during mammalian evolution; therefore, while only one DHRS4 gene is expressed in the mouse genome, the gene cluster consists of two (DHRS4 and DHRS4L1) and three (DHRS4, DHRS4L2, and DHRS4L1) copies in chimpanzees and humans, respectively. In this study, we explored the possible regulatory mechanism of the DHRS4 gene cluster in mammalian evolution by analyzing the promoter sequence, methylation of CpG islands, and RNA expression of the DHRS4 gene cluster in mice, chimpanzees, and humans by bioinformatics prediction, bisulfite sequencing PCR, and real-time reverse transcriptase-PCR. The results indicated that the DHRS4 gene was actively expressed in the three model species. The RNA level of DHRS4L1 was much lower than those of DHRS4 and DHRS4L2, and expressed lower homologous sequence identity to DHRS4 and DHRS4L2. DHRS4L2, the latest evolutionary copy of the DHRS4 gene in mammals, received a high promoter prediction score, and was the only copy of the DHRS4 gene cluster presenting hypermethylated CpG islands in the promoter region. An analysis of the relationship between the promoter characteristics and RNA expression of the DHRS4 gene cluster indicated that the development of CpG islands, in addition to the promoter sequence, during mammalian evolution could modulate the dose compensatory regulation of the copy number-varied DHRS4 gene cluster.
Assuntos
Ilhas de CpG , Metilação de DNA/genética , Evolução Molecular , Oxirredutases/genética , Animais , Ilhas de CpG/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Família Multigênica , Pan troglodytes , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Transcrição GênicaRESUMO
GABAA receptors are chloride channels in the brain that are activated by binding with g-aminobutyric acid (GABA). The cDNA sequences of GABAA receptor subunits from two strains of mice with different sensitivities to isoflurane were compared to identify nucleotide mutations. Included 80 mice from two strains with different sensitivities to isoflurane on C57BL/6 background. Forty mice were from an isoflurane-sensitive strain (S group) and 40 mice were from a resistant strain (R group). RNA was extracted from brains of the mice, and cDNA were reverse transcribed using AMV reverse transcriptase. The amplified products were processed, sequenced, and analyzed for differences between the two strains. Chi-square analysis was performed to compare differences in nucleotide mutation frequencies between the two strains. No differences were identified in the α1-6, ß2, ß3, or γ1-3 nucleotide sequences and no single nucleotide polymorphisms were found in the comparison with the GenBank sequence for the GABAA receptor subunit. A single nucleotide polymorphism (SNP) at the nucleotide position 462 (C/G) in the ß1 sequence was found. This SNP was observed in 5 mice from the sensitive strain and in 36 mice from the resistant strain. The Fischer exact test (P < 0.01) was used to compare two strains of mice for SNP in the cDNA sequence of the ß1 subunit. Additional studies are required to understand whether the GABAA receptor is a specific target of inhaled anesthetic action or whether the identified SNP affects the action of the volatile anesthetic.
Assuntos
Anestésicos Inalatórios/efeitos adversos , Resistência a Medicamentos/genética , Isoflurano/efeitos adversos , Receptores de GABA-A/genética , Anestésicos Inalatórios/administração & dosagem , Animais , Encéfalo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Isoflurano/administração & dosagem , Camundongos , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Bone fractures or bones subjected to open conduction and internal fixation are easily infected by bacteria; bacterial lipopolysaccharide (LPS) has been recognized as an important pathogenic factor affecting bone fracture healing. Therefore, the effect of LPS on bone metabolism is relevant for bone healing. In this study, we investigated the effect of LPS on the expression of Toll-like receptor (TLR)-4 (an LPS receptor) by using real-time quantitative PCR and western blotting. We also examined the regulatory role of LPS in osteoblast differentiation by measuring the ALP activity, matrix mineralization, and ALP, OCN, and Runx2 mRNA (essential factors affecting osteoblast differentiation) expression in LPS-treated mouse osteoblast MC3T3-E1 cells. We also evaluated the effect of TLR-4 on LPS-mediated inhibition of osteoblast differentiation using RNA interference. LPS promotes TLR-4 mRNA and protein expression in MC3T3-E1 cells (P < 0.05, P < 0.01 or P < 0.001), and inhibits osteoblast differentiation by downregulating matrix mineralization and ALP activity (P < 0.05, P < 0.01 or P < 0.001), and suppressing the expression ALP, OCN, and Runx2 mRNA in MC3T3-E1 cells (P < 0.05 or P < 0.01). Conversely, RNAi-mediated TLR-4 knockdown abrogates the LPS-mediated inhibition of osteoblast differentiation (P < 0.05 or P < 0.01). In summary, LPS was shown to inhibit osteoblast differentiation by suppressing the expression of ALP, OCN, and Runx2 in a TLR-4-dependent manner. The results of this study may provide insights into the signal pathway of LPS-induced bone loss or delayed bone fracture healing.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fraturas Ósseas/genética , Osteoblastos/metabolismo , Receptor 4 Toll-Like/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Fraturas Ósseas/tratamento farmacológico , Fraturas Ósseas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/administração & dosagem , Proteínas de Membrana/biossíntese , Camundongos , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genéticaRESUMO
APETALA2 plays critical roles in establishing meristem and organ identity during plant floral development. In this study, we obtained a CeAP2-like gene by using the mRNA differential display technique to analyze the wild type and a multitepal mutant of the orchid Cymbidium ensifolium. The full-length cDNA encoding the CeAP2-like transcription factor shows significant similarity to the cDNA of AP2 from Erycina pusilla and contains nucleotides complementary to miR172. Using a transient gene expression system of Arabidopsis protoplasts, we found that the accumulation of CeAP2-like protein and transcripts was negatively regulated by miR172, indicating this gene as a putative target of miR172. Northern blotting revealed that CeAP2-like is dominantly expressed in the sepals and petals of the wild-type flower, and shows low expression in the gynostemium. In contrast, the accumulation of CeAP2-like transcripts decreased significantly, especially in the central part of the mutant flower, corresponding to its abnormal petals and the absence of the gynostemium. Furthermore, we found an antagonistic expression pattern between CeAP2-like and AGAMOUS in the wild type, representing A- and C-class genes that specify floral organ fate. However, this antagonistic distribution was modified in the multitepal mutant, and both genes showed lower expression than that in the wild type. This result suggested that the balance between CeAP2-like and AGAMOUS activity was important for the regulation of floral patterning in C. ensifolium. This study represents the first report on a class A gene and its regulatory role for floral development in the orchid C. ensifolium.
Assuntos
Flores/genética , MicroRNAs/genética , Orchidaceae/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Orchidaceae/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Genome-wide re-sequencing of the Zhenshan 97 (ZS97) and Milyang 46 (MY46) parents of an elite three-line hybrid rice developed in China resulted in the generation of 9.91 G bases of data with an effective sequencing depth of 11.66x and 11.51x, respectively. Detection of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs), short insertions/deletions (InDels; 1-5 bp), and structural variations (SVs), which is an invaluable variation resource for genetic research and molecular marker-assisted breeding, was conducted by comparing whole-genome re-sequencing data. A total of 364,488 SNPs, 61,181 InDels and 6298 SVs were detected in ZS97 and 364,179 SNPs, 61,984 InDels and 6408 SVs were detected in MY46 compared to the 9311 reference sequence. Synteny analysis of the variation revealed a total of 77,013 identical and 181,737 different SNPs and 15,021 identical and 1205 different InDels between ZS97 and MY46, respectively. A total of 180 InDels 3-8 bp in length between ZS97 and MY46 were selected for experimental validation; 160 polymerase chain reaction products were efficiently separated on 6% non-denaturing polyacrylamide gels. Identification of genome-wide variation among the parents of the elite hybrid as well as the set of 160 polymerase chain reaction-based InDel markers will facilitate future genetic studies and the molecular breeding of hybrid rice.